EP4093426A1 - Modulation de vaisseaux lymphatiques dans une maladie neurologique - Google Patents

Modulation de vaisseaux lymphatiques dans une maladie neurologique

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Publication number
EP4093426A1
EP4093426A1 EP21706437.7A EP21706437A EP4093426A1 EP 4093426 A1 EP4093426 A1 EP 4093426A1 EP 21706437 A EP21706437 A EP 21706437A EP 4093426 A1 EP4093426 A1 EP 4093426A1
Authority
EP
European Patent Office
Prior art keywords
subject
neurological
therapeutic agent
antibody
genes
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21706437.7A
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German (de)
English (en)
Inventor
Jonathan Kipnis
Antoine LOUVEAU
Sandro DA MESQUITA
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UVA Licensing and Ventures Group
Original Assignee
University of Virginia Patent Foundation
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Filing date
Publication date
Application filed by University of Virginia Patent Foundation filed Critical University of Virginia Patent Foundation
Publication of EP4093426A1 publication Critical patent/EP4093426A1/fr
Pending legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1858—Platelet-derived growth factor [PDGF]
    • A61K38/1866—Vascular endothelial growth factor [VEGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer

Definitions

  • AD Alzheimer’s Disease
  • Mahnnow et al, 2006 the etiology of the amyloid pathology is poorly understood.
  • AD amyloid precursor protein
  • presenilins 1 and 2 drive the uncontrolled formation of amyloid beta
  • the brain’s pathological hallmarks of AD are intracellular neurofibrillary tangles and extracellular amyloid plaques, the latter being a product of the amyloidogenic processing of APP and the resulting deposition of amyloid beta in the brain parenchyma (Benilova et al, 2012; Hardy and Selkoe, 2002; Ittner and Gotz, 2011).
  • Increasing aggregation of diffusible amyloid beta peptides from the ISF and the CSF into toxic oligomeric intermediates and their accumulation in the brain parenchyma are believed to be precipitating factors for different neuroinflammatory abnormalities (Guillot-Sestier et al, 2015; Hong et al, 2016; Matarin et al, 2015), such as the formation of neurofibrillary tangles (Ittner and Gotz, 2011) and the pronounced neuronal dysfunction (Palop et al. , 2007 ; Sun el al.. 2009; Walsh el al. , 2002) in the AD brain.
  • Organs generally function less effectively with age. For example, skin becomes less elastic, muscle tone is lost, and heart function declines. Aging is a substantial risk factor for numerous neurological diseases, including neurodegenerative diseases and inflammatory neurological diseases.
  • Several embodiments herein relate generally to compositions, methods, and uses for modulating lymphatic vessels in the central nervous system. Modulating lymphatic vessels, in accordance with some embodiments, are used to treat, prevent, or ameliorate symptoms of neurological diseases.
  • the present invention provides compositions and methods for modulating lymphatic vessels of the central nervous system.
  • the compositions and methods are useful for treating, preventing, or ameliorating symptoms of neurological disease.
  • This application is related to PCT Application No. PCT/US2020/054390, fded on October 6, 2020, the entire contents of which are expressly incorporated herein by reference in their entirety.
  • the present disclosure provides a method of increasing clearance of molecules, such as proteins, in the central nervous system of a subject in need of treatment, inhibition, amelioration, reduction in symptoms, prevention, or delay in onset of a neurological disease.
  • the method includes administering an amount of a flow modulator to a subject, whereby the amount of flow modulator increases the diameter of a meningeal lymphatic vessel of the subject, thereby increasing fluid flow in the central nervous system of the subject; and administering a neurological therapeutic agent to the subject, whereby the clearance of molecules such as proteins in the central nervous system of the subject is increased.
  • the flow modulator is a VEGFR3 agonist or Fibroblast Growth Factor 2 (FGF2).
  • LEC lymphatic endothelial cell
  • Mg microglia
  • bBEC brain blood vascular cell
  • the alteration of gene expression is an increase in a level of gene expression of the one or more genes in Tables 2-29 as compared to a control level of gene expression of the one or more genes. In one embodiment, the alteration of gene expression is a decrease in a level of gene expression of the one or more genes in Tables 2-29 as compared to a control level of gene expression of the one or more genes. In one embodiment,
  • control level is a level of the gene expression of the one or more genes in Tables 2-29 in a healthy subject not having a neurological disease, or wherein the control level is an average level of gene expression of the one or more genes in Tables 2-29 in a population of healthy subjects not having a neurological disease.
  • control level is a level of the gene expression of the one or more genes in Tables 2-29 in an age-matched subject with intact and functional meningeal lymphatic vasculature and no underlying neurological disease, or wherein the control level is an average level of gene expression of the one or more genes in Tables 2-29 in a population of age-matched subjects with intact and functional mengigeal lymphatic vasculature and no neurological disease.
  • the level of gene expression of the one or more genes is increased by at least about 50%, 75%, 100%, 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, or more, as compared to the control level.
  • the level of gene expression of the one or more genes is increased by at least about 50% to about 5 -fold, about 2-fold to about 5-fold, about 3-fold to about 4-fold, about 50% to about 2-fold, about 50% to about 4-fold or about 75% to about 4-fold, as compared to the control level.
  • the level of gene expression of the one or more genes is increased by at least about 50%, 75%, 100%, 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, or more, as compared to the control level. In one embodiment, the level of gene expression of the one or more genes is decreased by at least about 50% to about 5 -fold, about 2-fold to about 5 -fold, about 3 -fold to about 4-fold, about 50% to about 2-fold, about 50% to about 4-fold or about 75% to about 4-fold, as compared to the control level.
  • control level is a level of the gene expression of the one or more genes in a healthy subject not having a neurological disease, or wherein the control level is an average level of gene expression of the one or more genes in a population of healthy subjects not having a neurological disease.
  • the method further comprises determining a level of gene expression of the one or more genes in the subject prior to administering the effective amount of the flow modulator and the effective amount of the neurological therapeutic agent to the subject. In one embodiment, the determination comprises obtaining a sample from the subject, processing the sample, and determining the level of gene expression. In one embodiment, the method further comprises determining a level of gene expression of the one or more genes in the subject after administering the effective amount of the flow modulator and the effective amount of the neurological therapeutic agent to the subject. In one embodiment, the determination comprises obtaining a sample from the subject, processing the sample, and determining the level of gene expression.
  • the method further comprises selecting a subject who would benefit from an increase in gene expression of the one or more genes in Tables 2-29 or a decrease in gene expression of the one or more genes in Tables 2-29.
  • the subject has a neurological disease, or is at risk for developing a neurological disease.
  • the method further comprises selecting a subject that has a neurological disease, or is at risk for developing a neurological disease.
  • the neurological disease is Alzheimer’s Disease (AD).
  • the subject has a risk factor for AD selected from the group consisting of: diploidy for apolipoprotein-E-epsilon-4 (apo-E-epsilon-4), a variant in apo-J, a variant in phosphatidylinositol-binding clathrin assembly protein (PICALM) , a variant in complement receptor 1 (CR3), a variant in CD33 (Siglee-3), or a variant in triggering receptor expressed on myeloid cells 2 (TREM2), age, familial AD, and a symptom of dementia; or a combination thereof.
  • apo-E-epsilon-4 diploidy for apolipoprotein-E-epsilon-4
  • PICALM phosphatidylinositol-binding clathrin assembly protein
  • CR3 complement receptor 1
  • Siglee-3 a variant in CD33
  • TREM2 myeloid cells 2
  • the flow modulator is a VEGFR3 agonist, said VEGFR3 agonist comprising VEGF-c.
  • the flow modulator e.g., VEGF-c
  • ICM intra- cistema magna
  • the neurological therapeutic agent comprises an antibody, or antigen-binding fragment thereof, that binds to amyloid beta.
  • the neurological therapeutic agent e.g., the antibody or antigen-binding fragment thereof, is administered systemically.
  • the method includes determining the subject to have the neurological disease, a risk factor therefor, or both.
  • the method includes determining the subject to have a risk factor for AD selected from the group consisting of: diploidy for apolipoprotein-E-epsilon-4 (apo-E-epsilon-4), a variant in apo-J, a variant in phosphatidylinositol- binding clathrin assembly protein (PICALM), a variant in complement receptor 1 (CR3), a variant in CD33 (Siglee-3), or a variant in triggering receptor expressed on myeloid cells 2 (TREM2), age, familial AD, a symptom of dementia, or a combination of any of the listed risk factors.
  • apo-E-epsilon-4 diploidy for apolipoprotein-E-epsilon-4
  • PICALM phosphatidylinositol- binding clathrin assembly protein
  • CR3 complement receptor 1
  • the disclosure provides a method of identifying a subject that has an enhanced risk of developing a neurological disease, comprising detecting an alteration in gene expression in one or more genes in Tables 2-29 in central nervous system prior to the onset of the neurological disease, thereby identifying the subject as having an enhanced risk of developing the neurological disease.
  • the alteration in gene expression is in brain lymphatic endothelial cells (LECs), brain myeloid cell (e.g., microglia (Mg)), infdtrating leukocyte and/or brain blood vascular cell (e.g., brain endothelial cells (bBECs)).
  • the alteration in gene expression is in immune cells in the brain of the subject.
  • the alteration in gene expression is in immune cells in brain cortices or meninges of the subject.
  • the gene is selected from the group consisting of Hexb, ApoE, H2-Aa, H2-AM, Cd74, H2-D1, and H- 2Kd.
  • the brain LECs or immune cells are obtained from a biopsy of deep cervical lymph nodes or peripheral blood from the subject.
  • the alteration in gene expression is in ear skin cells.
  • a method of identifying a subject that has an enhanced risk of developing neurological disease comprising detecting an increase in a number of immune cells in central nervous system of the subject prior to the onset of the neurological disease, thereby identifying the subject as having an enhanced risk of developing the neurological disease.
  • the increase in the number of immune cells is in brain cortices or meninges of the subject.
  • the immune cells are CD45high cells or H-2Kd expressing CD45int cells.
  • the immune cells are microglia or recruited lymphocytes from blood.
  • the immune cells are selected from the group consisting of B cells, CD4+ T cells, CD8+ T cells, and type 3 innate lymphoid cells (ILC3s). In one embodiment, the number of immune cells is determined by in vivo fluorescence imaging.
  • a method of identifying a subject that has an enhanced risk of developing a neurological disease comprising detecting one or more single nucleotide polymorphisms (SNPs) associated with one or more genes selected from the genes in Tables 2-29, thereby identifying the subject as having an enhanced risk of developing the neurological disease.
  • the SNP is associated with a gene that is highly expressed in a lymphatic endothelial cell.
  • the lymphatic endothelial cell is selected from the group consisting of a central nervous system lymphatic endothelial cell, a diaphragm lymphatic endothelial cell, and an ear skin endothelial cell.
  • the gene that is highly expressed in the lymphatic endothelial cell has an average expression in the top 2nd, 5th, 10th, or 25th percentile out of all genes. In one embodiment, the expression percentile is determined by R A-seq data. In one embodiment, the gene is selected from the group consisting of the genes listed in Figure 23.
  • the gene is selected from the group consisting of Dst, Hmcnl, Rgll, Prrc2c, Sft2d2, Itga6, Celfl, Sppl2a, Golim4, She, Abcal, Nfib, Akap9, Tmeml06b, Dlcl, AdamlO, Serinc5, Itgal, Ptprg, Fermt2, Efr3a, Parvb, Gsk3b, Pak2, Cd2ap, Egrl, and Ahnak.
  • the gene is selected from the group consisting of Frmd4a, Maf, Timp2, and Elmol.
  • the gene is selected from the group consisting of Crll, Clptml, Picalm, Psmal, Ssbp4, and Mef2c. In one embodiment, the gene is selected from the group consisting of Apoe Tspanl3, and Bsg.
  • the subject is a human subject.
  • the human subject is about 20 years old, about 30 years old, about 40 years old, about 50 years old, about 60 years old, about 70 years old, or about 80 years old.
  • the human subject has been previously identified to have a risk of developing neurological disease.
  • the human subject has been previously identified to have a risk of developing neurological disease by family history investigation or genetic screening.
  • the neurological disease is selected from the group consisting of AD (such as familial AD and/or sporadic AD), PD, cerebral edema, ALS, PANDAS, meningitis, hemorrhagic stroke, ASD, brain tumor (such as glioblastoma), epilepsy, Down’s syndrome, hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA- D), Familial Danish/British dementia, dementia with Lewy bodies (DLB), Lewy body (LB) variant of AD, multiple system atrophy (MSA), familial encephalopathy with neuroserpin inclusion bodies (FENIB), frontotemporal dementia (FTD), Huntington’s disease (HD), Kennedy disease/spinobulbar muscular atrophy (SBMA), dentatorubropallidoluysian atrophy (DRPLA); spinocerebellar ataxia (SCA) type I, SCA2, SCA3 (Machado-Josep
  • a method of reducing the risk or delaying the onset of developing a neurological disease in a subject comprising administering an effective amount of a neurological therapeutic agent to the central nervous system of the subject prior to the onset of the neurological disease, thereby reducing the risk of developing the neurological disease in the subject, wherein the subject is identified to have an enhanced risk of developing a neurodegenerative disease using a method described herein.
  • the method further comprises administering an effective amount of a flow modulator to the subject.
  • the neurological therapeutic agent reduces the number of immune cells in the brain.
  • a method of increasing clearance of a molecule from the central nervous system in a subject in need thereof comprising: administering an effective amount of a flow modulator to the subject by intra-cistema magna (ICM) injection, wherein the flow modulator increases the fluid flow in the central nervous system of the subject; and administering an effective amount of a neurological therapeutic agent to the subject by systemic administration, thereby increasing the clearance of the molecule from the central nervous system of the subject.
  • ICM intra-cistema magna
  • a method of reducing an aggregate of a protein or peptide in the central nervous system of a subject in need thereof comprising: administering an effective amount of a flow modulator to the subject by intra-cistema magna (ICM) injection, wherein the flow modulator increases the fluid flow in the central nervous system of the subject; and administering an effective amount of a neurological therapeutic agent to the subject by systemic administration, thereby reducing the aggregate of the protein or peptide in the subject.
  • ICM intra-cistema magna
  • a method of reducing a microglial inflammatory response in the central nervous system of a subject in need thereof comprising: administering an effective amount of a flow modulator to the subject by intra-cistema magna (ICM) injection, wherein the flow modulator increases the fluid flow in the central nervous system of the subject; and administering an effective amount of a neurological therapeutic agent to the subject by systemic administration, thereby reducing the microglial inflammatory response in the central nervous system of the subject.
  • ICM intra-cistema magna
  • a method of reducing neurite dystrophy in the central nervous system of a subject in need thereof comprising: administering an effective amount of a flow modulator to the subject by intra-cistema magna (ICM) injection, wherein the flow modulator increases the fluid flow in the central nervous system of the subject; and administering an effective amount of a neurological therapeutic agent to the subject by systemic administration, thereby reducing neurite dystrophy in the central nervous system of the subject.
  • ICM intra-cistema magna
  • a method of treating a neurological disease in a subject in need thereof comprising: administering an effective amount of a flow modulator to the subject by intra-cistema magna (ICM) injection, wherein the flow modulator increases the fluid flow in the central nervous system of the subject; and administering an effective amount of a neurological therapeutic agent to the subject by systemic administration, thereby treating the neurological disease in the subject.
  • the flow modulator comprises a VEGFR3 agonist, optionally wherein the VEGFR3 agonist comprises a VEGF-c.
  • the neurological therapeutic agent is an antibody, or antigen-binding fragment thereof, optionally wherein the antibody, or antigen-binding fragment thereof, is an amyloid beta antibody, or antigen-binding fragment thereof.
  • the amyloid beta antibody, or antigen-binding fragment thereof is selected from the group consisting of: bapineuzumab, gantenerumab, aducanumab, solanezumab, immunoglobulin, BAN2401, semorinemab, zagotenemab, crenezumab, and an antigen binding fragment thereof
  • the present invention provides a method of treating, inhibiting, ameliorating, reducing the symptoms of, preventing, or delaying the onset of a neurological disease.
  • the method includes administering an amount of a flow modulator to the subject in need, whereby the amount of the flow modulator increases the diameter of a meningeal lymphatic vessel of the subject; and administering a neurological therapeutic agent to the subject, wherein the neurological therapeutic agent is different from the flow modulator, thereby treating, inhibiting, ameliorating, reducing the symptoms of, preventing, or delaying the onset of the neurological disease.
  • the flow modulator is a VEGFR3 agonist or Fibroblast Growth Factor 2 (FGF2).
  • the neurological therapeutic agent is selected from the group consisting of a small molecule, a nucleic acid, a peptide, a protein, an antibody, a recombinant virus, a vaccine, and a cell.
  • the neurological therapeutic agent comprises a small molecule.
  • the small molecule is selected from the group consisting of Donepezil, Galantamine, Rivastigmine, Memantine, Fanabecestat, Atabecestat, Verubecestat, Elenbecestat, Semagacestat, Tarenflurbil, and Brexipiprazole.
  • the neurological therapeutic agent comprises an antibody, or an antigen binding fragment thereof, that specifically binds to a protein or a peptide that forms pathological aggregate.
  • the peptide or protein is selected from the group consisting of amyloid precursor protein, amyloid beta, fibrin, tau, apolipoprotein E (Apoe), alpha- synuclein, TDP43, and huntingtin.
  • the protein is amyloid beta and the antibody or the antigen binding fragment thereof is selected from the group consisting of: bapineuzumab, gantenerumab, aducanumab, solanezumab, immunoglobulin, BAN2401, semorinemab, zagotenemab, crenezumab, and the antigen binding fragment thereof.
  • the antibody or antigen binding fragment thereof that binds to amyloid beta comprises a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2, and a LCDR3 of any one of bapineuzumab, gantenerumab, aducanumab, solanezumab, immunoglobulin, BAN2401(Eisai), semorinemab, zagotenemab, crenezumab, or an antigen binding fragment thereof.
  • the protein is tau and the antibody or the antigen binding fragment thereof is selected from the group consisting of Gosuranemab, Armanezumab, and the antigen binding fragment thereof.
  • the antibody or antigen binding fragment thereof comprises a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2, and a LCDR3 of any one of Gosuranemab, Armanezumab, or the antigen binding fragment thereof.
  • the protein is alpha-synuclein and the antibody or the antigen binding fragment thereof is selected from the group consisting of BIIB054, PRX002/RG7935, prasinezumab, and the antigen binding fragment thereof.
  • the antibody or antigen binding fragment thereof comprises a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2, and a LCDR3 of any one of BIIB054, PRX002/RG7935, prasinezumab, or the antigen binding fragment thereof.
  • the protein is fibrin.
  • An exemplary antibody or the antigen binding fragment thereof is 5B8 as described in Ryu et al., Fibrin-targeting immunotherapy protects against neuroinflmmation and neurodegeneration, Nature Immunology 19, 1212-1223 (2016), incorporated herein by reference.
  • the antibody or antigen binding fragment thereof comprises a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2, and a LCDR3 of the 5B8 antibody.
  • the protein is apolipoprotein E (Apoe) and the antibody.
  • an exemplary antibody or the antigen binding fragment thereof is HAE4 as described in Liao et al., Targeting of nonlipidated, aggregated apoE with antibodies inhibits amyloid accumulation, J. of Clin. Invest., 128(5): 2144-2155, incorporated herein by reference.
  • the antibody or antigen binding fragment thereof comprises a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2, and a LCDR3 of the HAE4 antibody.
  • the flow modulator is VEGFR3 agonist, said VEGFR3 agonist comprising VEGF-c, and wherein the neurological therapeutic agent comprises an antibody that binds to amyloid beta.
  • the diameter of the meningeal lymphatic vessel is increased by at least
  • the central nervous system of the subject comprises amyloid beta plaques, and wherein administering the flow modulator in combination with the neurological therapeutic agent reduces the quantity of amyloid beta plaques.
  • the quantity of accumulated amyloid beta plaques is reduced by at least 5%.
  • at least some of the accumulated amyloid beta plaques are in the meninges of the subject’s brain.
  • the neurological disease is treated, inhibited, ameliorated, prevented, or delayed in onset, and/or wherein symptoms of the neurological disease are reduced.
  • the molecules to be cleared comprise a protein selected from the group consisting of amyloid beta, fibrin, tau, apolipoprotein E (Apoe), alpha synuclein, TDP43, and huntingtin.
  • a protein selected from the group consisting of amyloid beta, fibrin, tau, apolipoprotein E (Apoe), alpha synuclein, TDP43, and huntingtin.
  • the present invention provides method of reducing a quantity of aggregates of a protein or peptide in a subject having a neurological disease or a risk factor therefor.
  • the method includes determining the subject to have the neurological disease or the risk factor; administering a flow modulator to a meningeal space of the subject, whereby fluid flow in the central nervous system of the subject is increased; and administering a neurological therapeutic agent to the subject, wherein the neurological therapeutic agent is different from the flow modulator, thereby reducing the quantity of the aggregates of the protein or peptide in the subject.
  • the flow modulator is a VEGFR3 agonist or FGF2.
  • the protein or peptide is selected from the group consisting of amyloid beta, fibrin, tau, apolipoprotein E (Apoe), alpha synuclein, TDP43, and huntingtin.
  • the protein or peptide comprises amyloid beta and the aggregates comprise amyloid beta plaque.
  • the neurological therapeutic agent comprises an antibody that specifically binds to the protein or peptide.
  • the neurological therapeutic agent comprises a small molecule.
  • the aggregates of the protein or peptide are in the meninges of the subject’s brain.
  • the protein or peptide comprises amyloid beta and the aggregates comprise amyloid plaque.
  • the quantity of aggregates of the protein or peptide is reduced by at least 5%.
  • the protein or peptide comprises amyloid beta and the aggregates comprise amyloid plaque.
  • administering the flow modulator increases the diameter of a meningeal lymphatic vessel of the subject’s brain by at least 20%, thereby increasing fluid flow.
  • the flow modulator is VEGFR3 agonist or FGF2.
  • the VEGFR3 agonist is administered, and the VEGFR3 agonist comprising VEGF-c.
  • the subject has the neurological disease.
  • the VEGFR3 agonist is administered. In one embodiment, the
  • VEGFR3 agonist comprises VEGF-c
  • the neurological therapeutic agent comprises an antibody that binds to amyloid beta.
  • the VEGFR3 agonist is selected from the group consisting of: VEGF-c, VEGF-d, an analog, variant, or fragment thereof, or a combination of any of these.
  • the neurological therapeutic agent is administered to the central nervous system (CNS) of the subject.
  • the neurological therapeutic agent is administered to the meninges of the subject’s brain.
  • the flow modulator is administered by intra-cistema magna
  • the flow modulator is administered selectively to the meningeal space of the subject.
  • the flow modulator is administered to the subject by a route selected from the group consisting of: intrathecal administration, intraventricular administration, intraparenchymal administration, nasal administration, transcranial administration, contact with cerebral spinal fluid (CSF) of the subject, pumping into CSF of the subject, implantation into the skull or brain, contacting a thinned skull or skull portion of the subject with the flow modulator, expression in the subject of a nucleic acid encoding the flow modulator, or a combination of any of the listed routes.
  • the flow modulator is VEGFR3 agonist and/or FGF2.
  • the neurological therapeutic agent is administered selectively to the meningeal space of the subject.
  • the neurological therapeutic agent is administered to the subject by a route selected from the group consisting of: intrathecal administration, intraventricular administration, intraparenchymal administration, nasal administration, transcranial administration, contact with cerebral spinal fluid (CSF) of the subject, pumping into CSF of the subject, implantation into the skull or brain, contacting a thinned skull or skull portion of the subject with the neurological therapeutic agent, expression in the subject of a nucleic acid encoding the neurological therapeutic agent, intravenous infusion, or a combination of any of the listed routes.
  • a route selected from the group consisting of: intrathecal administration, intraventricular administration, intraparenchymal administration, nasal administration, transcranial administration, contact with cerebral spinal fluid (CSF) of the subject, pumping into CSF of the subject, implantation into the skull or brain, contacting a thinned skull or skull portion of the subject with the neurological therapeutic agent, expression in the subject of a nucleic acid encoding the neurological therapeutic agent, intravenous infusion, or a combination of any of the listed
  • the neurological therapeutic agent is administered to the subject systemically.
  • the neurological therapeutic agent is administered by intravenous infusion.
  • the neurological therapeutic agent is administered to the subject by the same route at the flow modulator. In another aspect, the neurological therapeutic agent is administered to the subject by a different route than the flow modulator.
  • the flow modulator and the neurological therapeutic agent are administered to the subject at the same time.
  • the flow modulator and the neurological therapeutic agent are administered to the subject in the same composition.
  • the flow modulator and the neurological therapeutic agent are administered to different locations of the subject.
  • the flow modulator and the neurological therapeutic agent are administered to the subject in different compositions.
  • the flow modulator and the neurological therapeutic agent are administered to the subject at different times.
  • the flow modulator is
  • the neurological disease comprises a proteinopathy.
  • the neurological disease comprises a tauopathy and/or amyloidosis.
  • the neurological disease is selected from the group consisting of: AD (such as familial AD and/or sporadic AD), PD, cerebral edema, AFS, PANDAS, meningitis, hemorrhagic stroke, ASD, brain tumor (such as glioblastoma), epilepsy, Down’s syndrome, hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D), Familial Danish/British dementia, dementia with Fewy bodies (DFB), Fewy body (FB) variant of AD, multiple system atrophy (MSA), familial encephalopathy with neuroserpin inclusion bodies (FENIB), frontotemporal dementia (FTD), Huntington’s disease (HD), Kennedy disease/spinobulbar muscular atrophy (SBMA), dentatorubropallidoluysian atrophy (DRPFA); spinocerebellar ataxia (SCA) type I, SCA2, SCA3 (Machado-J
  • AD such as familial AD
  • the neurological disease is an amyloidosis. In another embodiment, the neurological disease is Alzheimer’s disease. In still another embodiment, the neurological disease comprises familial AD and/or sporadic AD. In yet another embodiment, the neurological disease is familial AD and/or sporadic AD.
  • increasing fluid flow in the central nervous system of the subject comprises increasing a rate of perfusion of fluid throughout an area of the subject’s brain, and/or increasing a rate of perfusion of fluid through the subject’s central nervous system and/or increasing a rate of perfusion out of the subject’s central nervous system.
  • increasing the fluid flow in the CNS increases clearance of soluble molecules in the brain of the subject.
  • the fluid comprises cerebral spinal fluid (CSF), interstitial fluid (ISF), or both.
  • the neurological therapeutic agent is administered in an amount effective to treat, inhibit, ameliorate, reduce the symptoms of, prevent, or delay the onset of the neurological disease.
  • the fluid comprises cerebral spinal fluid (CSF), interstitial fluid
  • the neurological therapeutic agent is selected from the group consisting of: bapineuzumab, gantenerumab, aducanumab, solanezumab, crenezumab, pepinemab, ozanezumab, AT-1501, BIIB054, and PRX002.
  • the present invention provides a composition or product combination.
  • the composition or product combination includes a flow modulator; and a neurological therapeutic agent that is different from the flow modulator.
  • the flow modulator is VEGFR3 agonist or Fibroblast Growth Factor 2 (FGF2).
  • FGF2 Fibroblast Growth Factor 2
  • the neurological therapeutic agent is selected from the group consisting of a small molecule, a nucleic acid, a peptide, a protein, an antibody, a recombinant virus, and a cell.
  • the neurological therapeutic agent comprises a small molecule.
  • the small molecule is selected from the group consisting of Donepezil, Galantamine, Rivastigmine, Memantine, Fanabecestat,
  • Atabecestat Verubecestat, Elenbecestat, Semagacestat, Tarenflurbil, and Brexipiprazole.
  • the neurological therapeutic agent comprises an antibody that binds to a protein that forms pathological aggregate.
  • the peptide or protein is selected from the group consisting of amyloid precursor protein, amyloid beta, fibrin, tau, apolipoprotein E (Apoe), alpha-synuclein, TDP43, and huntingtin.
  • the protein is amyloid beta and the antibody or the antigen binding fragment thereof is selected from the group consisting of: bapineuzumab, gantenerumab, aducanumab, solanezumab, immunoglobulin, BAN2401, semorinemab, zagotenemab, crenezumab, and the antigen binding fragment thereof.
  • the antibody or antigen binding fragment thereof that binds to amyloid beta comprises a HCDR1, a HCDR2, a HCDR3, a FCDR1, a FCDR2, and a FCDR3 of any one of bapineuzumab, gantenerumab, aducanumab, solanezumab, immunoglobulin, BAN2401(Eisai), semorinemab, zagotenemab, crenezumab, or the antigen binding fragment thereof.
  • the protein is tau and the antibody or the antigen binding fragment thereof is selected from the group consisting of Gosuranemab, Armanezumab, and the antigen binding fragment thereof.
  • the antibody or antigen binding fragment thereof comprises a HCDR1, a HCDR2, a HCDR3, a FCDR1, a FCDR2, and a FCDR3 of any one of Gosuranemab, Armanezumab, or the antigen binding fragment thereof.
  • the protein is alpha-synuclein and the antibody or the antigen binding fragment thereof is selected from the group consisting of BIIB054 (Biogen),
  • the antibody or antigen binding fragment thereof comprises a HCDR1, a HCDR2, a HCDR3, a FCDR1, a FCDR2, and a FCDR3 of any one of BIIB054 (Biogen), PRX002/RG7935 (Roche), prasinezumab (Roche), or the antigen binding fragment thereof.
  • the protein is fibrin.
  • An exemplary antibody or the antigen binding fragment thereof is 5B8 as described in Ryu et al., Fibrin-targeting immunotherapy protects against neuroinflmmation and neurodegeneration, Nature Immunology 19, 1212-1223 (2016).
  • the antibody or antigen binding fragment thereof comprises a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2, and a LCDR3 of the 5B8 antibody.
  • the protein is apolipoprotein E (Apoe).
  • An exemplary antibody or the antigen binding fragment thereof is HAE4 as described in Liao et al., Targeting of nonlipidated, aggregated apoE with antibodies inhibits amyloid accumulation, J. of Clin. Invest., 128(5): 2144- 2155.
  • the antibody or antigen binding fragment thereof comprises a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2, and a LCDR3 of the HAE4 antibody.
  • the VEGFR3 agonist comprises VEGF-c. In one embodiment, the
  • VEGFR3 agonist comprises VEGF-c
  • the neurological therapeutic agent comprises an antibody that binds to amyloid beta.
  • the neurological therapeutic agent is selected from the group consisting of bapineuzumab, gantenerumab, aducanumab, solanezumab, crenezumab, pepinemab, ozanezumab, AT-1501, BIIB054, and PRX002.
  • the VEGFR3 agonist is selected from the group consisting of:
  • VEGF-c VEGF-d
  • the flow modulator is in an amount effective to increase fluid flow in the central nervous system of the subject.
  • the flow modulator is an amount effective to diameter of a meningeal lymphatic vessel of the subject’s brain by at least 20%, thereby increasing fluid flow.
  • composition or product combination further includes a pharmaceutically acceptable carrier.
  • composition or product combination is formulated for a route of administration selected from the group consisting of: intrathecal administration, intraventricular administration, intraparenchymal administration, nasal administration, transcranial administration, contacting cerebral spinal fluid (CSF) of the subject, pumping into CSF of the subject, implantation into the skull or brain, contacting a thinned skull or skull portion of the subject with the flow modulator and the neurological therapeutic agent.
  • a route of administration selected from the group consisting of: intrathecal administration, intraventricular administration, intraparenchymal administration, nasal administration, transcranial administration, contacting cerebral spinal fluid (CSF) of the subject, pumping into CSF of the subject, implantation into the skull or brain, contacting a thinned skull or skull portion of the subject with the flow modulator and the neurological therapeutic agent.
  • the present invention provides composition or product combination.
  • the composition or product combination includes a first nucleic acid that encodes the flow modulator of any aspects described herein, wherein the flow modulator is a polypeptide; and a neurologic therapeutic agent.
  • the flow modulator is VEGFR3 agonist or FGF2.
  • the neurologic therapeutic agent is a second nucleic acid encoding the protein, the peptide, or the antibody of any aspects described herein, wherein the protein, the peptide, or the antibody is not the flow modulator.
  • the first nucleic acid and second nucleic acid are DNA or RNA.
  • the first nucleic acid and second nucleic acid are a DNA, and the DNA is on an expression vector.
  • the expression vector is a plasmid or a viral vector.
  • the expression vector is an adeno- associated viral vector.
  • the first nucleic acid and the second nucleic acid are on the same polynucleotide molecule. In another embodiment, the first nucleic acid and the second nucleic acid are on different polynucleotide molecule.
  • the present invention provides a pharmaceutical composition.
  • the pharmaceutical composition includes the composition or the product combination of any aspects described herein, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is formulated for a route of administration selected from the group consisting of: intrathecal administration, intraventricular administration, intraparenchymal administration, nasal administration, transcranial administration, contacting cerebral spinal fluid (CSF) of the subject, pumping into CSF of the subject, implantation into the skull or brain, contacting a thinned skull or skull.
  • CSF cerebral spinal fluid
  • the present invention provides the composition or product combination or the pharmaceutical composition of any aspects described herein for use in treating a neurological disease or disorder.
  • the use is in in treating a proteinopathy, such as a tauopathy and/or amyloidosis.
  • the use is in in treating a neurological disease selected from the group consisting of: AD (such as familial AD and/or sporadic AD), PD, cerebral edema, ALS, PANDAS, meningitis, hemorrhagic stroke, ASD, brain tumor (such as glioblastoma), epilepsy, Down’s syndrome, hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D), Familial Danish/British dementia, dementia with Lewy bodies (DLB), Lewy body (LB) variant of AD, multiple system atrophy (MSA), familial encephalopathy with neuroserpin inclusion bodies (FENIB), frontotemporal dementia (FTD), Huntington’s disease (HD), Kennedy disease/spinobulbar muscular atrophy (SBMA), dentatorubropallidoluysian atrophy (DRPLA); spinocerebellar ataxia (SCA) type I, SCA2, SCA3 (Machad
  • the flow modulator is a polypeptide, and wherein the flow modulator is administered by administering an effective amount of a first nucleic acid that encoding the polypeptide.
  • the neurological therapeutic agent is a second nucleic acid encoding the peptide, the protein, or the antibody of any one of claims 1-70, wherein the protein, the peptide, or the antibody is not the flow modulator.
  • the first nucleic acid and second nucleic acid are DNA or RNA. In one embodiment, the first nucleic acid and second nucleic acid are a DNA, and the DNA is on an expression vector. In another embodiment, the expression vector is a plasmid or a viral vector. In still another embodiment, the expression vector is an adeno-associated viral vector. [0084] In one aspect, the first nucleic acid and the second nucleic acid are on the same polynucleotide molecule. In another aspect, the first nucleic acid and the second nucleic acid are on different polynucleotide molecule.
  • a method of increasing clearance of molecules (such as proteins) in the central nervous system of a subject in need of treatment, inhibition, amelioration, reduction in symptoms, prevention, or delay in onset of a neurological disease is described.
  • the method can comprise administering an amount of VEGFR3 agonist or Fibroblast Growth Factor 2 (FGF2) to a meningeal space of the subject, whereby the amount of VEGFR3 agonist or FGF2 increases the diameter of a meningeal lymphatic vessel of the subject, thereby increasing fluid flow in the central nervous system of the subject.
  • the method can comprise administering a neurological therapeutic agent to the central nervous system of the subject.
  • the clearance of molecules such as proteins in the central nervous system of the subject can be increased.
  • the method further comprises determining the subject to have the neurological disease, a risk factor therefor, or both. In some embodiments, the method further comprises determining the subject to have a risk factor for AD selected from the group consisting of: diploidy for apolipoprotein-E-epsilon- 4 (apo-E-epsilon-4), a variant in apo-J, a variant in phosphatidylinositol-binding clathrin assembly protein (PICALM) , a variant in complement receptor 1 (CR3), a variant in CD33 (Siglee-3), or a variant in triggering receptor expressed on myeloid cells 2 (TREM2), age, familial AD, a symptom of dementia, or a combination of any of the listed risk factors.
  • apo-E-epsilon-4 diploidy for apolipoprotein-E-epsilon- 4
  • PICALM phosphatidylinositol-binding clathrin assembly protein
  • a method of treating, inhibiting, ameliorating, reducing the symptoms of, preventing, or delaying the onset of a neurological disease is described.
  • the method can comprise administering an amount of VEGFR3 agonist or Fibroblast Growth Factor 2 (FGF2) to a meningeal space of the subject in need, whereby the amount of VEGFR3 agonist or FGF2 increases the diameter of a meningeal lymphatic vessel of the subject.
  • the method can comprise administering a neurological therapeutic agent for the neurological disease to the subject, wherein the neurological therapeutic agent is different from the VEGFR3 or FGF2.
  • the method can treat, inhibit, ameliorate, reduce the symptoms of, prevent, or delay the onset of the neurological disease.
  • the neurological therapeutic agent comprises an antibody that specifically binds to amyloid beta.
  • the amyloid beta antibody is selected from the group consisting of: bapineuzumab, gantenerumab, aducanumab, solanezumab, and crenezumab.
  • the antibody that binds to amyloid beta comprises a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2, and a LCDR3 of any one of bapineuzumab, gantenerumab, aducanumab, solanezumab, or crenezumab.
  • the VEGFR3 agonist is administered, said VEGFR3 agonist comprising VEGF-c, and the neurological therapeutic agent comprises an antibody that binds to amyloid beta.
  • the diameter of the meningeal lymphatic vessel is increased by at least 20%.
  • the central nervous system of the subject comprises amyloid beta plaques, and wherein administering the VEGFR3 agonist or FGF2 in combination with the neurological therapeutic agent reduces the quantity of amyloid beta plaques.
  • the quantity of accumulated amyloid beta plaques is reduced by at least 5%.
  • the accumulated amyloid beta plaques are in the meninges of the subject’s brain.
  • the neurological disease is treated, inhibited, ameliorated, prevented, or delayed in onset, and/or wherein symptoms of the neurological disease are reduced.
  • the molecules comprise amyloid beta.
  • method of reducing a quantity of accumulated amyloid beta plaques in a subject having a neurological disease or a risk factor therefor is described.
  • the method can comprise determining the subject to have the neurological disease or the risk factor.
  • the method can comprise administering a VEGFR3 agonist or FGF2 to a meningeal space of the subject, so that fluid flow in the central nervous system of the subject is increased.
  • the method can comprise administering a neurological therapeutic agent to the subject, wherein the neurological therapeutic agent is different from the VEGFR3 or FGF2.
  • the method can reduce the quantity of accumulated amyloid beta plaques in the subject.
  • the neurological therapeutic agent comprises an antibody that specifically binds to amyloid beta.
  • the accumulated amyloid beta plaques are in the meninges of the subject’s brain.
  • the quantity of accumulated amyloid beta plaques is reduced by at least 5%.
  • the VEGFR3 agonist or FGF2 increases the diameter of a meningeal lymphatic vessel of the subject’s brain by at least 20%, thereby increasing fluid flow.
  • the VEGFR3 agonist is administered, said VEGFR3 agonist comprising VEGF-c.
  • the subject has the neurological disease.
  • the VEGFR3 agonist is administered.
  • the VEGFR3 agonist can comprises VEGF-c
  • the neurological therapeutic agent can comprise an antibody that binds to amyloid beta.
  • the VEGFR3 agonist can be selected from the group consisting of: VEGF-c, VEGF-d, an analog, variant, or fragment thereof, or a combination of any of these.
  • the neurological therapeutic agent is administered to the central nervous system (CNS) of the subject.
  • CNS central nervous system
  • the neurological therapeutic agent can be administered to the meninges of the subject’s brain.
  • the VEGFR3 agonist and/or FGF2 is administered selectively to the meningeal space of the subject.
  • the VEGFR3 agonist and/or FGF2 is administered to the subject by a route selected from the group consisting of: intrathecal administration, intraventricular administration, intraparenchymal administration, nasal administration, transcranial administration, contact with cerebral spinal fluid (CSF) of the subject, pumping into CSF of the subject, implantation into the skull or brain, contacting a thinned skull or skull portion of the subject with the VEGFR3 agonist or FGF2, expression in the subject of a nucleic acid encoding the VEGFR3 agonist or FGF2, or a combination of any of the listed routes.
  • the neurological therapeutic agent is administered selectively to the meningeal space of the subject.
  • the neurological therapeutic agent is administered to the subject by a route selected from the group consisting of: intrathecal administration, intraventricular administration, intraparenchymal administration, nasal administration, transcranial administration, contact with cerebral spinal fluid (CSF) of the subject, pumping into CSF of the subject, implantation into the skull or brain, contacting a thinned skull or skull portion of the subject with the neurological therapeutic agent, expression in the subject of a nucleic acid encoding the neurological therapeutic agent, intravenous infusion, or a combination of any of the listed routes.
  • CSF cerebral spinal fluid
  • the neurological therapeutic agent is administered to the subject by intravenous infusion. In some embodiments, for any of the methods described herein, the neurological therapeutic agent is administered to the subject by the same route at the VEGFR3 agonist and/or FGF2. In some embodiments, for any of the methods described herein, the neurological therapeutic agent is administered to the subject by a different route than the VEGFR3 agonist and/or FGF2. In some embodiments, for any of the methods described herein, the VEGFR3 agonist or FGF2 and the neurological therapeutic agent are administered to the subject at the same time.
  • the VEGFR3 agonist or FGF2 and the neurological therapeutic agent are administered to the subject in the same composition. In some embodiments, for any of the methods described herein, the VEGFR3 agonist or FGF2 and the neurological therapeutic agent are administered to different locations of the subject. In some embodiments, for any of the methods described herein, the VEGFR3 agonist or FGF2 and the neurological therapeutic agent are administered to the subject in different compositions. In some embodiments, for any of the methods described herein, the VEGFR3 agonist and FGF2 and the neurological therapeutic agent are administered to the subject at different times.
  • the neurological disease comprises a proteinopathy. In some embodiments, for any of the methods described herein, the neurological disease comprises a tauopathy and/or amyloidosis. In some embodiments, for any of the methods described herein, the neurological disease is selected from the group consisting of: AD (such as familial AD and/or sporadic AD), PD, cerebral edema, ALS, PANDAS, meningitis, hemorrhagic stroke, ASD, brain tumor (such as glioblastoma), epilepsy, Down’s syndrome, hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D), Familial Danish/British dementia, dementia with Lewy bodies (DLB), Lewy body (LB) variant of AD, multiple system atrophy (MSA), familial encephalopathy with neuroserpin inclusion bodies (FENIB), frontotemporal dementia (FTD), Huntington’s disease
  • AD familial AD and/or sporadic AD
  • the neurological disease is an amyloidosis. In some embodiments, for any of the methods described herein, the neurological disease is Alzheimer’s disease. In some embodiments, for any of the methods described herein, the neurological disease comprises familial AD and/or sporadic AD. In some embodiments, for any of the methods described herein, the neurological disease is familial AD and/or sporadic AD.
  • increasing fluid flow in the central nervous system of the subject comprises increasing a rate of perfusion of fluid throughout an area of the subject’s brain, and/or increasing a rate of perfusion of fluid through the subject’s central nervous system and/or increasing a rate of perfusion of fluid out of the subject’s central nervous system.
  • the method can comprise increasing a rate of perfusion out of the subject’s central nervous system.
  • increasing the fluid flow in the CNS increases clearance of soluble molecules in the brain of the subject.
  • the fluid comprises cerebral spinal fluid (CSF), interstitial fluid (ISF), or both.
  • the neurological therapeutic agent is administered in an amount effective to treat, inhibit, ameliorate, reduce the symptoms of, prevent, or delay the onset of the neurological disease.
  • the fluid comprises cerebral spinal fluid (CSF), interstitial fluid (ISF), or both.
  • the neurological therapeutic agent is selected from the group consisting of: bapineuzumab, ganteneramab, aducanumab, solanezumab, crenezumab, pepinemab, ozanezumab, AT-1501, BIIB054, and PRX0002.
  • a composition or product combination is described.
  • the composition or product combination can comprise a VEGFR3 agonist or FGF2.
  • the composition or product combination can comprise a neurological disease therapeutic agent that is different from the VEGFR3 agonist or FGF2.
  • the neurological therapeutic agent comprises an antibody that binds to amyloid beta.
  • the VEGFR3 agonist comprises VEGF-c, and wherein the neurological therapeutic agent comprises an antibody that binds to amyloid beta.
  • the antibody that binds to amyloid beta is selected from the group consisting of bapineuzumab, ganteneramab, aducanumab, solanezumab, and crenezumab.
  • the antibody that binds to amyloid beta comprises a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2, and a LCDR3 of any one of bapineuzumab, gantenerumab, aducanumab, solanezumab, or crenezumab.
  • the VEGFR3 agonist comprises VEGF-c, and wherein the neurological therapeutic agent comprises an antibody that binds to amyloid beta.
  • the neurological therapeutic agent is selected from the group consisting of bapineuzumab, gantenerumab, aducanumab, solanezumab, crenezumab, pepinemab, ozanezumab, AT-1501, BIIB054, and PRX0002.
  • the VEGFR3 agonist is selected from the group consisting of: VEGF-c, VEGF-d, an analog, variant, or fragment thereof, or a combination of any of these.
  • the VEGFR3 agonist or FGF2 is an amount effective to increase fluid flow in the central nervous system of the subject. In the composition or product combination of some embodiments, the VEGFR3 agonist or FGF2 is an amount effective to diameter of a meningeal lymphatic vessel of the subject’s brain by at least 20%, thereby increasing fluid flow.
  • the composition or product combination of some embodiments further comprises a pharmaceutically acceptable carrier.
  • the composition or product combination is formulated for a route of administration selected from the group consisting of: intrathecal administration, intraventricular administration, intraparenchymal administration, nasal administration, transcranial administration, contact cerebral spinal fluid (CSF) of the subject, pumping into CSF of the subject, implantation into the skull or brain, contacting a thinned skull or skull portion of the subject with the agent, and expression in the subject of a nucleic acid encoding the VEGFR3 agonist, FGF2, and/or neurological therapeutic agent.
  • the composition or product combination of some embodiments is for use in treating a neurological disease or disorder.
  • composition or product combination of some embodiments is for use in treating a proteinopathy, such as a tauopathy and/or amyloidosis.
  • the composition or product combination of some embodiments is for use in treating a neurological disease selected from the group consisting of: AD (such as familial AD and/or sporadic AD), PD, cerebral edema, ALS, PANDAS, meningitis, hemorrhagic stroke, ASD, brain tumor (such as glioblastoma), epilepsy, Down’s syndrome, hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D), Familial Danish/British dementia, dementia with Fewy bodies (DEB), Fewy body (FB) variant of AD, multiple system atrophy (MSA), familial encephalopathy with neuroserpin inclusion bodies (FENIB), frontotemporal dementia (FTD), Huntington’s disease (HD), Kennedy disease/spinobulbar muscular atrophy (SBMA), dentatorubro
  • FIGs. 1A-1G are a series of microscope images and graphs showing effects of the flow modulator VEGF-c and the neurological therapeutic agent amyloid beta antibody on the meninges and meningeal vasculature of adult 5xFAD mice in accordance with some embodiments.
  • FIG. 1A depicts the age of the of 5xFAD mice and treatment regimen.
  • FIG. IB shows representative images of the meningeal whole-mounts of 5xFAD mice treated with different combinations of mIgG2a or monoclonal anti-amyloid beta antibody (“ABETA Mabl”) with AAVl-CMV-eGFP or AAVl-CMV-mVEGF-C. Meninges were stained for FYVE-1 and CD31; scale bar, 1 mm; inset, 300 pm.
  • ABETA Mabl monoclonal anti-amyloid beta antibody
  • the units for the Y-axis are percentage of field of view (“%FOV”).
  • FIGs. 2A-2M are a series of microscope images and graphs showing effects of the flow modulator VEGF-c and the neurological therapeutic agent amyloid beta antibody on amyloid beta protein and plaques in adult 5xFAD mice in accordance with some embodiments.
  • FIG. 2A shows representative images of the brain sections of these mice. Brain sections were stained for Ab and with DAPI; scale bar, 2mm.
  • FIGs. 2B-2M show plaque density (number of plaques per mm 2 ) (FIGs. 2B-2E), average size (pm 2 ) (FIGs. 2F-2I) and coverage (% of brain section) (FIGs. 2J-2M) in particular brain regions (cortex and amygdala, FIGs. 2B, 2F and 2J; hippocampus, FIGs. 2C, 2G and 2K; thalamus and hypothalamus, FIGs. 2D, 2H and 2L) or in the whole brain section (FIGs. 2E, 21 and 2M).
  • FIGs. 3A-3G are a series of graphs and microscope images showing effects of the flow modulator VEGF-c and the neurological therapeutic agent amyloid beta antibody on behavior and amyloid beta plaques of aged APPswe mice in accordance with some embodiments. Behaviors assessed include open field, novel location recognition, and contextual fear conditioning.
  • FIG. 3A depicts the age of the of APPswe mice and treatment regimen.
  • FIG. 3B depicts total distance, velocity and time in center of the arena (% of total time) in the open field test.
  • FIG. 3C depicts time investigating one of the object location (% of total time investigating objects) in the training trial and time investigating the novel object location (% of total time investigating) in the novel location recognition test.
  • FIG. 3D depicts time freezing (% of total time) in the context trial and in cued trial of the contextual fear condition test.
  • FIG. 3E show representative images of the brain sections of APPswe mice treated with anti-Abeta antibody (ABETA Mabl) and with AAVl-CMV-eGFP or AAVl-CMV-mVEGF-C. Brain sections were stained for Ab and with DAPI; scale bar, 1mm.
  • FIGs. 4A-4Q show meningeal immune response in 3 months-old 5xFAD mice and
  • FIG. 4A is representative flow cytometry dot plots showing gating strategy for
  • CD64 + macrophages pre-gated in singlets CD45 + live cells
  • CD19 + B cells pre-gated in CD64 neg cells
  • CD11o + MHO-II w811 dendritic cells DCs, pre-gated in CD19 neg cells
  • FIG. 4B is representative flow cytometry dot plots showing gating strategy for gdT cells (pre-gated in singlets CD45 + live cells), TCRp neg NKl.l + cells (NK cells, pre-gated in TCR neg cells), TCRp + NKl.l + cells (NKT cells, pre-gated in TCRp + cells) and CD4 + and CD8 + T cells (pre gated in TCRp + NKl.l 1168 cells).
  • FIGs. 4C-4K show number of total CD45 + live (FIG. 4C), CD64 + macrophages
  • FIG. 4D is histograms showing the expression levels of PD-1 in the fluorescence minus one (FMO) sample (grey) or in concatenated meningeal immune cell populations from WT and 5xFAD groups. Cells considered to be positive for PD-1 are demarcated in the different histograms.
  • FIGs. 4M-4Q show frequencies of PD-1 -expressing gdT cells (FIG. 4M), NK cells
  • FIG. 4N NKT cells (FIG. 40), CD4 + T cells (FIG. 4P) and CD8 + T cells (FIG. 4Q) in each group.
  • FIGs. 5A-5R show compromised meningeal lymphatic function is observed in
  • FIG. 5A is representative images of meningeal whole mounts from 5-6- and 13-14 months-old 5xFAD mice, stained for CD31, LYVE-1 and Ab (stained with the D54D2 antibody; scale bar, 2 mm; inset scale bar, 500 pm).
  • FIG. 5B is a scheme depicting the compartmentalization of the meningeal whole mount for quantification of LYVE-1 and Ab coverage.
  • FIGs. 5C-5E are graphs showing the coverage by LYVE-1 + CD31 + vessels as a percentage of the region of interest (% of ROI) at the superior sagittal sinus (SSS) (FIG. 5C), transverse sinus and confluence of sinuses (TS/COS) (FIG. 5D), and petrosquamosal and sigmoid sinuses (PS S/S S) (FIG. 5E).
  • SSS superior sagittal sinus
  • TS/COS transverse sinus and confluence of sinuses
  • PS S/S S petrosquamosal and sigmoid sinuses
  • FIG. 5F shows that lymphatic endothelial cells (LECs) were isolated from the brain meninges of 5xFAD mice and WT littermate controls at 6 months of age, total RNA was extracted and sequenced.
  • LECs lymphatic endothelial cells
  • FIG. 5G is principal component analysis (PCA) plot showing segregation between
  • FIG. 5H is a heatmap of top 200 differentially expressed genes.
  • FIG. 51 shows gene set obtained by functional enrichment of differentially expressed genes in meningeal LECs from 5xFAD mice.
  • FIG. 5J shows that human LECs were incubated with 100 nM synthetic scrambled human Ab42 peptides (scramble) or synthetic human monomeric/dimeric Ab42 for 24h or 72h, total RNA was extracted and sequenced.
  • FIG. 5K is PCA plot showing segregation between the different samples.
  • FIG. 5L is a heatmap of top 200 differentially expressed genes.
  • FIG. 5M is gene sets obtained by functional enrichment of differentially expressed genes in human LECs incubated with Ab42 for 72h, when compared to scramble 72h.
  • 5N shows representative images of skull caps with attached meningeal layers after photo-ablation.
  • Adult 2 months-old WT mice were injected (i.c.m.) with Visudyne (5 pL) followed by a transcranial photoconversion step (Vis./photo.) to ablate meningeal lymphatic vessels.
  • Control mice were injected with Visudyne without photoconversion (Vis.).
  • mice were injected with 5 pL of a suspension of fluorescent microspheres (1 pm in diameter) into the CSF and 15 minutes later the lymphatic vessel afferent to the deep cervical lymph node (dCLN) was imaged by in vivo stereomicroscopy.
  • Representative images of skull cap show microspheres and lymphatic vessels stained for LYVE-1 around the confluence of sinuses (COS) and transverse sinus (TS) at the dorsal brain meninges or around the sigmoid (SS) and petrosquamosal (PSS) sinuses at the basal brain meninges (scale bars, 500 pm).
  • COS sinuses
  • TS transverse sinus
  • SS sigmoid
  • PSS petrosquamosal
  • FIG. 5Q is representative frames showing microspheres flowing through the lymphatic vessel afferent to the dCLN, or cumulative sphere tracking (for 20 sec), in mice with intact or ablated meningeal lymphatic vessels.
  • FIGs. 6A-6F show functional enrichment analysis of differentially expressed genes in meningeal LECs from 5xFAD mice and in human LECs incubated with AP 42 .
  • FIG. 6A is a heatmap of top 50 differentially expressed genes in WT and 5xFAD
  • FIGs. 6B and 6C are Exocytosis (G0:0006887) and Phospholipase D signaling pathway (KEGG:mmu04072) gene sets obtained by functional enrichment analysis, with corresponding differentially expressed genes.
  • FIG. 6D is a heatmap of top 50 differentially expressed genes in scramble or AP 42 at
  • FIGs. 6E and 6F are adherens junctions (KEGG:hsa04520) and Phospholipase D signaling pathway (KEGG:hsa04072) gene sets obtained by functional enrichment analysis, with corresponding differentially expressed genes.
  • FIGs. 7A-7E show mass cytometry gating strategy, marker expression levels, unsupervised clustering, and meningeal immune cell numbers.
  • FIG. 7A shows representative mass cytometry dot plots depicting gating strategy used to select CD45 + live cells used in further high-dimensional analysis. Meningeal single-cell suspensions were obtained from 5xFAD mice at 5-6 months and 11-12 months and processed for mass cytometry.
  • FIG. 7B shows final heatmaps of the median marker expression values for each immune cell cluster identified using Rphenograph. Median marker expression values are indicated by color intensity depicted in the scale bar.
  • FIG. 7C is t-distributed stochastic neighbor embedding (tSNE) plots showing unsupervised clustering of CD45 + live immune cells.
  • FIG. 7D is number of total CD45 + live meningeal leukocytes.
  • FIG. 7E is numbers of different meningeal immune cell clusters showing a statistically significant increase in B cells, CD4 + T cells, CD8 + T cells, type 3 innate lymphoid cells (ILC3s) and an undefined cell population isolated from the meninges of 5xFAD mice at 11-12 months of age.
  • FIGs. 8A-8C show specificity of an anti-Abeta antibody (ABETA Mabl) and kinetics of brain Ab recognition upon i.c.m. or i.v. injections.
  • FIG. 8A is representative images of brain sections from 4 months-old 5xFAD mice and WT littermate controls that were incubated without primary antibody (ab), with murine IgG2a isotype control (anti-fluorescein), anti-Abeta antibody murine IgG2a monoclonal antibody (against human Ab protofibrils, ABETA Mabl) or a commercially available rabbit anti-human Ab (D54D2 clone, optimal for immunofluorescence staining). Images show Ab and DAPI staining (scale bar, 1 mm).
  • FIG. 8B presents images of different regions of the brain of 5xFAD mice, 1-hour post injection of anti-Abeta antibody (1 pig/ pL) into the CSF (5 pL, i.c.m.) or into the blood (100 pL, i.v.).
  • FIG. 8C presents images of different regions of the brain of 5xFAD mice 24 hours post injection of anti-Abeta antibody into the CSF (i.c.m.) or into the blood (i.v.). Images in FIG. 8B and FIG. 8C panels show astrocyte endfeet and glia limitans stained for Aquaporin 4 (AQP4), Ab stained with Amylo-Glo RTD and anti-Abeta antibody staining (scale bar, 200 pm). Data is representative of 2 independent experiments.
  • FIGs. 8D-8N show pharmacological ablation of the dorsal meningeal lymphatic vessels in 5xFAD mice dampens Ab plaque clearance by anti-Abeta antibody.
  • FIG. 8D shows that adult 4-5 months-old female 5xFAD mice were injected (i.c.m.) with Visudyne (5 pL) plus photoconversion (Vis./photo.) or Visudyne without photoconversion (Vis.).
  • mice were injected (i.c.m.) with anti-Abeta antibody (murine antibody against Ab protofibrils, ABETA Mabl) or murine IgG2a (mIgG2a) as a control (both at 1 pig/ pL).
  • anti-Abeta antibody murine antibody against Ab protofibrils, ABETA Mabl
  • mIgG2a murine IgG2a
  • Injection of anti-Abeta antibody or mIgG2a was repeated two weeks later.
  • Another lymphatic vessel ablation step was performed, followed by two injections with anti-Abeta antibody or mIgG2a.
  • FIG. 8E is representative images of brain sections from 5xFAD mice stained for Ab and with DAPI (scale bar, 1 mm).
  • FIGs. 8F and 8G are graphs showing number of Ab plaques per mm 2 , average size of
  • FIG. 8H is representative stereomicroscopy images of lymphatic vessels stained for
  • LYVE-1 around the confluence of sinuses (COS) and transverse sinus (TS) at the dorsal brain meninges or around the sigmoid (SS) and petrosquamosal (PSS) sinuses at the basal brain meninges still attached to the skull cap (scale bars, 500 pm).
  • FIGs. 8I-8L are graphs showing LYVE-1 + vessel total length (in mm) (FIG. 81) and branching points (FIG. 8J) in the dorsal meninges and total length (in mm) (FIG. 8K) and branching points (FIG. 8L) in the basal meninges.
  • FIGs. 9A-9K show repeated delivery of anti-Abeta antibody (ABETA Mabl) into the
  • CSF of 5xFAD mice reduces brain Ab plaque load.
  • FIG. 9A shows that adult 5xFAD mice (3 months-old) were injected (i.c.m.) with either mIgG2a (5 pL at 1 pg/pL) or anti-Abeta antibody (5 pL at 0.1 or 1 pg/pL). Injections were repeated another three times, every two weeks, as shown in the scheme.
  • FIG. 9B is representation of different brain regions considered for the quantification of Ab, namely hippocampus, cortex/striatum/amygdala and thalamus/hypothalamus.
  • FIG. 9C is representative images of brain sections from the different groups stained for Ab and with DAPI (scale bar, 2 mm).
  • FIGs. 9D-9K are quantification of average size of Ab plaques (pm 2 ) and coverage of
  • FIGs. 9D and 9E Ab (% of section) in the hippocampus (FIGs. 9D and 9E), cortex/striatum/amygdala (FIGs. 9F and 9G), thalamus/hypothalamus (FIGs. 9H and 91) and in the whole brain section (FIGs. 9J and 9K).
  • FIGs. 10A-10K show that compromising meningeal lymphatic function in 5xFAD mice limits brain Ab clearance by ABETA Mabl and modulates neuritic dystrophy, microglial activation and fibrinogen levels.
  • FIG. 10A shows that adult 3-3.5 months-old male 5xFAD mice were injected
  • mice received intraperitoneal (i.p.) injections of ABETA Mab 1 (a murine antibody against Ab protofibrils) or the control murine IgG (mlgG) antibodies, each at a dose of 40 mg/kg.
  • Antibodies were injected weekly for a total of four weeks. Additional steps of meningeal lymphatic vessel ablation or control interventions were followed by four weekly injections with antibodies. Mice were tested in the open field and Morris water maze.
  • FIG. 10B show representative images of brain sections from 5xFAD mice stained for
  • FIGs. lOC-lOF are graphs showing number of Ab plaques per mm 2 of brain section
  • FIG. IOC average size of Ab plaques (pm 2 ) in ABETA Mabl cohort (FIG. 10D), , coverage by Ab plaques (FIG. 10E ) and coverage by LAMPU dystrophic neurites (FIG. 10F) (as % of brain section) in each group.
  • FIG. 10G shows representative images of the brain cortex stained for Ab (stained with Amilo-Glo), fibrinogen (grey), IBA and CD68 (scale bar, 100 pm).
  • FIGs. 10H-10K are graphs showing the coverage by IBA1 + cells (% of field) (FIG.
  • FIGs. 11A-11G show that meningeal lymphatic dysfunction leads to anxious-like behavior and worsened spatial learning and memory in 5xFAD mice.
  • FIG. 11A shows that adult 5xFAD mice with intact or ablated meningeal lymphatics and treated with ABETA Mabl or control mlgG antibodies were tested in the open field arena and in the Morris water maze.
  • FIGs. 11B-12D are graphs showing total distance (in centimeters) (FIG. 12B), velocity (in centimeters per second) (FIG. 11C) and percentage of time in the center of the open field arena (% of total time) (FIG. 11D).
  • FIGs. 11E-11G are graphs showing latency to platform in acquisition (in seconds)
  • FIG. HE percentage of time in the platform quadrant in the probe trial
  • FIG. 11F percentage of time in the platform quadrant in the probe trial
  • FIG. 11G latency to platform in reversal
  • FIGs. 12A-12F show impairing meningeal lymphatic drainage affects the access of anti-Abeta antibody (ABETA Mabl) to Ab plaques in the brain parenchyma.
  • 5xFAD mice (5 months- old) with intact or ablated meningeal lymphatic vasculature were injected (i.c.m.) with 5 pL of anti- Abeta antibody (at 1 pig/ pL) .
  • mice were transcardially perfused and the brain was collected for analysis.
  • FIGs. 12A and 12B are images of ten different regions of the brain of 5xFAD mice from the Visudyne (Vis.) or Visudyne plus photoconversion (Vis./photo.) groups showing blood vessels stained for CD31, Ab stained with Amylo-Glo RTD and anti-Abeta antibody staining (scale bar, 200 pm).
  • FIGs. 12C and 12D are graphs with colocalization between CD31 and anti-Abeta antibody (% of CD31 signal occupied by anti-Abeta antibody) in each brain region (1 to 10) (FIG. 12C) or presented as the average of all regions (FIG. 12D).
  • FIGs. 12E and 12F are graphs with quantifications of colocalization between Ab aggregates and anti-Abeta antibody (% of Ab signal occupied by anti-Abeta antibody) in each brain region (1 to 10) (FIG. 12E) or presented as the average of all regions (FIG. 12F).
  • FIGs. 13A-13U show impairing meningeal lymphatic drainage in 5xFAD mice affects microglial gene expression.
  • FIG. 13A shows that myeloid cells were sorted (by fluorescence activated cell sorting, FACS) from the brain cortex of 4 months-old 5xFAD mice injected with Visudyne alone (Vis.) or Visudyne followed by transcranial photoconversion (Vis./photo.). Transcriptome of sorted live CD45 + Ly6G neg CDl lb + cells pooled from 3 mice per group was analyzed by single-cell RNA-seq (scRNA-seq).
  • FIG. 13B depicts unsupervised clustering and t-distributed stochastic neighbor embedding (tSNE) representation of four distinct clusters of microglia.
  • tSNE stochastic neighbor embedding
  • FIG. 13C shows frequency of microglia (% of total cells) from each cluster was similar when comparing the Vis. and Vis./photo. groups.
  • FIG. 13D is a heatmap with genes involved in the transition from homeostatic to disease-associated microglia phenotypes, depicting the homeostatic (clusters 1 and 2), Trem2 independent (cluster 3) and Trem2 dependent (cluster 4) signatures within each cell. Cells were grouped by cluster and genes were grouped by signature. Scale shows mean-centered, log -normalized expression values.
  • FIGs. 13E and 13F are violin plots showing Hexb expression levels e) in microglia from each cluster (FIG. 13E) and all microglia (FIG. 13F).
  • FIGs. 13G and 13H are violin plots showing Apoe expression levels in microglia from each cluster (FIG. 13G) and all microglia (FIG. 13H).
  • FIGs. 13I-13L are violin plots showing expression of the major histocompatibility complex II genes Cd74 (FIG. 131), H2-D1 (FIG. 13J), H2-Aa (FIG. 13K) and H2-Ab I (FIG. 13L).
  • Data in FIGs. 13B-13L resulted from the analysis of 402 microglia in the Vis. group and 249 microglia in the Vis./photo. group (from sequenced cells sorted from the cortices of 3 mice per group); Wilcoxon Rank-Sum test with Bonferroni’s multiple comparisons test was used in FIGs. 13E-13L.
  • FIG. 13M shows that flow cytometry was performed using cell suspensions from the brain cortex of 4 months-old 5xFAD mice injected with Visudyne alone (Vis.) or Visudyne followed by transcranial photoconversion (Vis./photo.).
  • FIG. 13N is representative flow cytometry dot plots showing gating strategy for live
  • CD45'" sl 'CD l 1 b" es (lymphoid cells)
  • CD45'" sl 'CD l lb ⁇ (recruited and/or activated myeloid cells)
  • CD45 int CDl lb + (microglia).
  • FIGs. 130-13Q are frequencies of CD45 mt CD 1 lb + (FIG. 130), CD45 Mgh CD 1 lb +
  • FIG. 13P and CD45 hlgh CDl lb neg (FIG. 13Q) cells isolated from the brain cortex of 5xFAD mice from the Vis. and Vis./photo. groups.
  • FIG. 13R is histograms showing the expression levels of H-2Kd in the fluorescence minus one (FMO) sample (grey) or in concatenated immune cell populations from the Vis. and Vis./photo. groups. Cells considered to be positive for H-2Kd are demarcated in the different histograms.
  • FIGs. 13S-13U are geometric mean fluorescence intensity (gMFI) values relative to
  • FIGs. 14A-14I show that meningeal lymphatic vessel ablation precludes brain Ab plaque clearance by ABETA Mabl administered into the CSF.
  • FIG. 14A shows that adult 4-4.5 months-old male 5xFAD mice were injected
  • FIG. 14B shows representative images of meningeal whole mounts stained for CD31
  • LYVE-1 and Ab stained with the D54D2 antibody; scale bar, 2 mm).
  • FIG. 14C is a graph showing the coverage by Ab as a percentage of the meningeal whole mount.
  • FIG. 14D shows representative images of brain sections from 5xFAD mice stained for Ab (stained with the D54D2 antibody) and with DAPI (scale bar, 2 mm).
  • FIGs. 14E-14G are graphs showing number of Ab plaques per mm 2 of brain section
  • FIG. 14E average size of Ab plaques (pm 2 ) (FIG. 14F)and total coverage of Ab plaques (% of brain section) (FIG. 14G) in each group.
  • FIG. 14H is representative inset showing an example of a Prussian blue focus in a brain tissue section of a 5xFAD mouse (scale bar, 100 pm).
  • FIG. 141 is a graph showing the quantifications of Prussian blue foci per brain section in each group.
  • FIGs. 15A-15R show combination therapy with mVEGF-C and anti-Abeta antibody
  • ABSETA Mabl induces meningeal lymphangiogenesis and boosts brain Ab plaque clearance.
  • FIG. 15A shows that adult 5xFAD mice were injected with 5 pL (i.c.m.) of AAV1 expressing enhanced green fluorescent protein (eGFP) or murine VEGF-C (mVEGF-C), under the cytomegalovirus (CMV) promoter (each at 10 12 GC/pL), in combination with either mIgG2a or anti- Abeta antibody (each at 1 pg/pL) as indicated in the scheme.
  • eGFP enhanced green fluorescent protein
  • mVEGF-C murine VEGF-C
  • CMV cytomegalovirus
  • FIG. 15B show representative images of brain sections stained for Ab (stained with the D54D2 antibody) and with DAPI (scale bar, 2 mm).
  • FIG. 15C is graph showing coverage of Ab as percentage of brain section in each group.
  • FIG. 15D shows representative images from the brain cortex stained for Ab (stained with the Amilo-Glo), CD68 and IBA1 (scale bar, 50 pm).
  • FIGs. 15E-15G are graphs showing the coverage by IBA1 + cells (% of field) (FIG.
  • FIG. 15H is representative stereomicroscopy images of lymphatic vessels stained for
  • LYVE-1 around the transverse sinus (TS) at the dorsal brain meninges or around the sigmoid (SS) and petrosquamosal (PSS) sinuses at the basal brain meninges still attached to the skull cap (scale bars,
  • FIG. 15K is representative images of meningeal whole mounts stained for CD31 and
  • LYVE-1 scale bar, 1 mm; inset scale bar, 300 pm).
  • FIGs. 15L and 15M are graphs showing b) coverage of CD3 l + LYVE-l neg vessels (% of meningeal whole mount) (FIG. 15L) and c) branching points and coverage of LYVE-1 + vessels (% of meningeal whole mount) (FIG. 15M). Results in FIGs.
  • FIG. 15N is representative images of brain sections from 5xFAD mice stained for Ab and with DAPI (scale bar, 2 mm).
  • FIGs. 150-15R are graphs showing the coverage of Ab (% of brain region/section) in the hippocampus (FIG. 150), cortex/striatum/amygdala (FIG. 15P), thalamus/hypothalamus (FIG. 15Q) and the whole brain section (FIG. 15R).
  • FIG. 15S is representative images from the brain cortex stained for Ab, LAMP-1 and
  • FIGs. 15T and 15U are graphs showing the coverage (% of field) by LAMP-1 + dystrophic neurites (FIG. 15T) and Fibrinogen (FIG.15U) in cortical vasculature.
  • FIGs. 16A-16N show viral-mediated expression of mVEGF-C induces transcriptomic changes in aged meningeal LECs and improves the efficacy of anti-Abeta antibody treatment (ABETA Mabl) in aged AD transgenic mice.
  • FIG. 16A shows that aged WT mice (20-24 months of age) were injected with 2 pL
  • AAV1 expressing eGFP or mVEGF-C under the CMV promoter (each at 10 13 GC/pL).
  • mice were transcardially perfused, skull caps were collected, meninges harvested and LECs were sorted by FACS for bulk RNA-seq.
  • FIG. 16B is PCA plot showing segregation between eGFP and mVEGF-C meningeal
  • FIG. 16C is a heatmap of top 50 differentially expressed genes. Color scale bar represents expression values for each sample as standard deviations from the mean across each gene in FIG. 19C.
  • FIG. 16D is volcano plot showing the significantly down-regulated and up-regulated genes between meningeal LECs from the mVEGF-C and eGFP groups.
  • FIG. 16E shows that aged J20 mice (14-16 months-old) were injected with 5 pL
  • AAV1 expressing eGFP or mVEGF-C (each at 10 12 GC/pL) in combination with either mIgG2a or anti-Abeta antibody (each at 1 pg/pL) as indicated in the scheme.
  • FIG. 16F is representative images of brain sections from J20 mice stained for Ab and with DAPI (scale bar, 1 mm).
  • FIG. 16J shows aged APPswe mice (26-30 months-old) were injected with 5 pL
  • AAV1 expressing eGFP or mVEGF-C (each at 10 12 GC/pL) in combination with either mIgG2a or anti-Abeta antibody (each at 1 pg/pL) as indicated in the scheme.
  • FIG. 16K is representative images of brain sections from APPswe mice stained for
  • FIGs. 17A-17C show genes associated with increased risk for Alzheimer’s disease and other neurological disorders are highly expressed in mouse lymphatic endothelial cells.
  • FIG. 17A is pie charts showing the proportion of genes associated with higher risk for Alzheimer’s disease, Parkinson’s disease, Schizophrenia, Autism spectrum disorder and Multiple sclerosis, for which the average expression across all LEC RNA-seq datasets was in the top 2 nd , 5 th , 10 th , or 25 th percentile out of all genes.
  • FIG. 17B is heatmaps showing the log2-normalized expression values (depicted in color scale bar) for disease-associated genes whose average expression values fall within the top 2 nd percentile of genes expressed across RNA-seq datasets obtained from LECs isolated from diaphragm, ear skin and meninges of 2-3 months (m.) old mice (Louveau, A. et al. CNS lymphatic drainage and neuroinflammation are regulated by meningeal lymphatic vasculature. Nat Neurosci 21, 1380-1391, (2016)), from meninges of 2-3 or 20-24 months-old mice (Da Mesquita, S. et al. Functional aspects of meningeal lymphatics in ageing and Alzheimer's disease.
  • FIG. 17C is gene sets obtained by functional enrichment of 25 th percentile disease- associated genes expressed across the different LEC RNA-seq datasets.
  • FIGs. 18A-18D show genes associated with increased risk for Alzheimer’s disease are highly expressed in cultured human LECs and brain blood endothelial cells.
  • FIG. 18A is pie charts showing the proportion of genes associated with higher risk for Alzheimer’s disease for which the average expression in cultured human LEC RNA-seq datasets was in the top 2 nd , 5 th , 10 th , or 25 th percentile out of all genes.
  • FIG. 18B is a heatmap showing the log2 -normalized expression values (depicted in color scale bar) for disease-associated genes whose average expression values fall within the top 2 nd percentile of genes expressed in the RNA-seq datasets obtained from cultured human LECs.
  • FIG. 18C is pie charts showing the proportion of genes associated with higher risk for Alzheimer’s disease for which the average expression in brain capillary endothelial cells (ECs) 1, capillary ECs 2, arterial ECs and venous ECs (obtained from the scRNA-seq dataset published by Vanlanderwijck et al. (Vanlanderwijck, M. et al. A molecular atlas of cell types and zonation in the brain vasculature. Nature 554, 475-480, (2016))) was in the top 2 nd , 5 th , 10 th , or 25 th percentile out of all genes.
  • FIG. 18D is a heatmap showing the log2-normalized expression values (depicted in color scale bar) for disease-associated genes whose average expression values fall within the top 2 nd percentile of genes expressed in capillary ECs 1, capillary ECs 2, arterial ECs and venous ECs.
  • FIG. 18E shows that the transcriptome of myeloid cells (live CD45 + Ly6GXDl lb + cells) sorted from the brain cortex of 5.5 month-old 5xFAD mice was analyzed by single-cell RNA- seq (scR A-seq).
  • the graph shows the unsupervised clustering and tSNE representation of four distinct clusters of microglia (Mg).
  • FIG. 18F depicts pie charts showing the proportion of Alzheimer’s disease- associated gene single nucleotide polymorphisms, for which the average expression was in the top 2 nd , 5 th , 10 th , or 25 th percentile out of all genes in each microglial cluster.
  • FIG. 18G is a heatmap showing the expression values for Alzheimer’s disease- associated genes whose average expression values fall within the top 2 nd percentile of all genes expressed in each microglial cluster.
  • FIGs. 18C and 18D resulted from the analysis of a scRNA-seq dataset published by Vanriewijck el al n : data in FIGs. 18E-18G resulted from the scRNA-seq analysis of 651 microglia; in FIGs. 18D and 18G, scale bars represent log2-normalized expression values; see FIGs. 17A-17C and FIG. 19 for more details and data.
  • FIG. 19 is a Venn diagram showing overlap in Alzheimer’s SNP associated genes in the top 10 th percentile for EC, LEC and microglia.
  • FIGs. 20A - 20C depict heatmaps showing the study of the differences in lymphatic endothelial cell constitution.
  • FIG. 20A depicts heatmaps showing that meningeal lymphatic endothelial cells have signatures that distinguish them from diaphragm and skin lymphatics.
  • FIG. 20B depicts heatmaps showing the changes observed in meningeal lymphatics of young and old mice.
  • FIG. 20C depicts heatmaps showing the changes observed in gene expression level in hippocampal cells after blockage of meningeal lymphatics.
  • FIG. 21 depicts heatmaps and graphs showing Alzheimer’s disease-specific pathways impacted in LEC cultures treated with Ab in a temporal manner.
  • FIG. 22 depicts heatmaps showing the identification of differentially expressed genes
  • This invention is based upon, at least partially, the unexpected discovery that the treatment with flow modulators, e.g., VEGF-c, in combination with a neurological therapeutic agent, e.g, an amyloid-b antibody, can synergize to reduce protein aggregates, e.g., amyloid-b plaques, in the central nervous system, e.g., brain.
  • Flow modulators can increase flow for example, by increasing the diameter of a meningeal lymphatic vessel of the subject, by increasing the quantity of meningeal lymphatic vessels of the subject, and/or by increasing drainage through meningeal lymphatic vessels of the subject.
  • fluid flow in the central nervous system of the subject can be increased.
  • Neurological therapeutic agents interact with a target, e.g.
  • the flow modulators may facilitate the removal of the “end product” of the interaction between neurological therapeutic agents and the target, e.g., the complex formed between a pathological protein and an antibody, by various mechanisms as described in detail herein, e.g., increasing the drainage of the “end product” to certain specific sites, e.g., deep cervical lymph nodes, thereby improving the treatment of neurodegenerative disease.
  • the flow modulator may facilitate access of the neurological therapeutic agent to its target.
  • a treatment combining a flow modulator described herein with a therapeutic agent described herein may improve the extent of the desired effect on the pathology of the neurological disease, e.g., reduction of pathological protein aggregates, reduction of inflammation at the site of aggregation, etc., as compared to a treatment with the therapeutic agent alone or as compared to a treatment with the flow modulator alone.
  • compositions and methods using one or more flow modulators, e.g., VEGF-c in combination with a neurological therapeutic agents to increase the therapeutic effect over the single agent alone.
  • the central nervous system was viewed as being immune privileged, and was believed to lack a classical lymphatic drainage system.
  • a lymphatic system is present in meningeal spaces, and functions in draining macromolecules, immune cells, and debris from the central nervous system (CNS).
  • CNS central nervous system
  • modulating lymphatic vessels to increase flow in accordance with some embodiments herein can synergize with neurological therapeutic agents to alleviate symptoms of neurological diseases, for example proteinopathies such as tauopathies and/or amyloidoses (e.g., AD), including cognitive symptoms, and accumulation of amyloid beta plaques.
  • neurological therapeutic agents for example proteinopathies such as tauopathies and/or amyloidoses (e.g., AD)
  • proteinopathies such as tauopathies and/or amyloidoses (e.g., AD)
  • cognitive symptoms e.g., and accumulation of amyloid beta plaques.
  • methods, compositions, and uses for treating, preventing, inhibiting, or ameliorating symptoms of neurological diseases, for example proteinopathies such as tauopathies and/or amyloidoses (e.g., AD) are described.
  • the neurological diseases can be associated with increased concentration and/or the accumulation of macromolecules, cells, and debris in the CNS (for example, AD, which is associated with the accumulation of amyloid beta plaques).
  • the methods, compositions, and uses can increase drainage by lymphatic vessel, and thus increase flow in CSF and ISF.
  • Several embodiments herein are particularly advantageous because they include one, several or all of the following benefits: (i) increased flow in the CNS; (ii) decreased accumulation of macromolecules, cells, or debris in the CNS (for example, decreased accumulation of amyloid beta); and (iii) maintenance of or improvement in motor and/or cognitive function (for example memory function) in a subject suffering from, suspected of having, and/or at risk for a neurological disease (such as dementia in a neurological disease such as AD).
  • a neurological disease such as dementia in a neurological disease such as AD.
  • meningeal lymphatic vessels mediate drainage in the CNS, and that impaired meningeal lymphatic function impacts brain homeostasis. See, e.g., PCT Pub. No. 2017/210343, which is incorporated by reference herein in its entirety. Characteristics of meningeal lymphatic vessels are described for example, in PCT Pub. No. 2017/210343 at Example 13. Immune cells such as T cells and dendritic cells accumulate in the meningeal lymphatic vessels (See, e.g., PCT Pub. No. 2017/210343 at Examples 14-21). Impairing meningeal vessels significantly decreases drainage into deep cervical lymph nodes, and impact immune cell size and coverage, and inhibits immune cell migration (PCT Pub. No. 2017/210343 at Examples 2 and 24-25).
  • flow shall be given its ordinary meaning and shall also refer to a rate of perfusion through an area of the central nervous system of a subject. Flow in some embodiments, can be measured as a rate at which a label or tracer in CSF perfuses through a particular area of the central nervous system (see, e.g., FIGs. 3A-3J of WO2017/210343). As such, flow can be compared between two subjects or two sets of conditions by ascertaining how quickly an injected label or tracer perfuses throughout a particular area or volume of the brain and/or other portion of the CNS.
  • flow modulators shall be given its ordinary meaning and shall also broadly refer to classes of compositions that can increase or decrease the passage of substances into and out of meningeal lymphatic vessels, and thus can modulate flow in CSF and ISF, and/or, can modulate immune cell migration within, into, and out of the meningeal lymphatic vessels.
  • flow modulators increase the diameter of meningeal lymphatic vessels, which increases drainage, resulting in increased flow in the CSF and ISF. In some embodiments, flow modulators increase the number of meningeal lymphatic vessels, thus increasing net drainage, resulting in increased flow in the CSF and ISF.
  • suitable flow modulators for increasing flow include, but are not limited to, VEGFR3 agonists, for example VEGF-c and VEGF-d, and Fibroblast Growth Factor 2 (FGF2), and functional fragments, variants, analogs, and mimetics of these molecules.
  • VEGFR3 agonists for example VEGF-c and VEGF-d
  • FGF2 Fibroblast Growth Factor 2
  • a flow modulator e.g. ,
  • VEGFR3 agonists, or FGF2 comprises or consists essentially of a polypeptide or protein that comprises a modification, for example a glycosylation, PEGylation, or the like.
  • a composition or composition for use in accordance with methods and uses described herein comprises or consists essentially of one or more flow modulators (e.g., VEGFR3 agonists, or FGF2), one or more neurological therapeutic agents (e.g., amyloid beta antibody), and a pharmaceutically acceptable diluent or carrier.
  • flow modulators e.g., VEGFR3 agonists, or FGF2
  • neurological therapeutic agents e.g., amyloid beta antibody
  • a pharmaceutically acceptable diluent or carrier examples of suitable pharmaceutically acceptable carriers and formulations are described in “Remington: The Science and Practice of Pharmacy” 22nd Revised Edition, Pharmaceutical Press, Philadelphia, 2012, which is hereby incorporated by reference in its entirety.
  • the composition comprises or consists essentially of a unit dose of a flow modulator effective for increasing flow of CNS fluids, increasing clearance of molecules in the CNS, or reducing a quantity of accumulated amyloid beta plaques in accordance with methods or uses as described herein.
  • the composition comprises, or consists essentially of a single unit dose of flow modulator effective for increasing flow, increasing clearance reducing accumulated amyloid beta plaques, reducing immune cell migration, or reducing inflammation.
  • the effective amount of flow modulator is about 0.00015 mg/kg to about 1.5 mg/kg (including any other amount or range contemplated as a therapeutically effective amount of a compound as disclosed herein), is less than about 1.5 mg/kg (including any other range contemplated as a therapeutically effective amount of a compound as disclosed herein), or is greater than 0.00015 mg/kg (including any other range contemplated as a therapeutically effective amount of a compound as disclosed herein).
  • VEGFR3 also known as FLT4
  • FLT4 is a receptor tyrosine kinase, and its signaling pathway has been implicated in embryonic vascular development, and adult lymphangiogenesis.
  • VEGFR3 Upon binding of ligand, VEGFR3 dimerizes, and is activated through autophosphorylation. It is shown herein that VEGFR3 agonists are a class of flow modulators that increase the diameter of meningeal lymphatic vessels, and which increase drainage and the flow of CSF and ISF in accordance with some embodiments herein (see Examples 4-6, FIGs. 26, 27A-27D, 28A, and 28C of WO 2017/210343).
  • VEGFR3 agonists are suitable for methods, compositions, and uses for treating, ameliorating, reducing the symptoms of, or preventing neurological diseases associated with accumulation of molecules in the brain, for example proteinopathies as described herein (e.g., tauopathies and/or amyloidoses such as AD), in accordance with some embodiments herein.
  • a flow modulator comprises, consists of, or consists essentially of a VEGFR3 agonist.
  • VEGFR3 agonist in accordance with methods, compositions, and uses herein can be understood in terms of its ability to increase meningeal vessel diameter, by its ability to increase flow of CSF or ISF, or by its ability to treat, ameliorate, or prevent (by its ability to increase clearance of substances such as proteins from the CNS, for example amyloid beta), symptoms of a neurological disease such as proteinopathies as described herein (e.g., tauopathies and/or amyloidoses such as AD), for example quantities of beta-amyloid plaques or measurements of cognitive function.
  • a neurological disease such as proteinopathies as described herein (e.g., tauopathies and/or amyloidoses such as AD), for example quantities of beta-amyloid plaques or measurements of cognitive function.
  • an effective amount of VEGFR3 agonist increases meningeal vessel diameter by at least about 2%, for example, at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%, including ranges between any two of the listed values.
  • an effective amount of VEGFR3 agonist increases flow of the CSF or ISF by at least about 2%, for example, at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%, including ranges between any two of the listed values.
  • Example VEGFR3 agonists suitable for methods, uses, and compositions in accordance with some embodiments herein include the polypeptides VEGF-c and VEGF-d, the amino acid sequences of which are shown in Table 1, below, as well as variants and analogs of VEGF-c and/or VEGF-d.
  • VEGF-c in accordance with some embodiments herein has been demonstrated to increase the diameters of meningeal lymphatic vessels, and to increase drainage, CSF and ISF flow, and clearance in the CNS. See Example 4 of WO2017/210343.
  • a VEGFR3 agonist comprises, consists of, or consists essentially of VEGF-c.
  • a VEGFR3 agonist comprises, consists of, or consists essentially of VEGF-d.
  • VEGF-c and VEGF-d together agonize VEGFR3, and can be provided in a single composition, or in separate compositions.
  • a VEGFR3 agonist comprises, consists of, or consists essentially of an analog, variant, or functional fragment, such as a mutant, ortholog, fragment, or truncation of VEGF-c or VEGF-d, for example a polypeptide comprising, or consisting essentially of an amino acid sequence having at least about 80% identity to SEQ ID NO: 1 or 2 or 3, for example at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity, including ranges between any two of the listed values.
  • exogenous nucleotides encoding a VEGFR3 agonist such as VEGF-c
  • a nucleotide encoding VEGF-c or VEGF-d as describe herein is expressed in a subject in order to administer the VEGFR3 agonist to a subject.
  • an exogenous vector such as a retroviral, lentiviral, adenoviral, or adeno-associated viral vector comprising or consisting essentially of a nucleic acid encoding a VEGFR agonist as described here can be inserted into a host nucleic acid of the subject (for example in the genome of a somatic cell of the subject).
  • the vector further comprises transcriptional machinery to facilitate the transcription of the nucleic acid encoding the VEGFR agonist, for example, a core promoter, transcriptional enhancer elements, insulator elements (to insulate from repressive chromatin environments), and the like.
  • the VEGFR3 agonist comprises a modification, for example a glycosylation, PEGylation, or the like.
  • a composition for use in accordance with the methods described herein comprises the VEGFR3 agonist (e.g., VEGF-c and/or VEGF-d), and a pharmaceutically acceptable diluent or carrier.
  • the flow modulator comprises or consists essentially of
  • FGF2 Fibroblast Growth Factor 2
  • FGF2 can increase drainage (and flow) of CSF or ISF in meningeal lymphatic vessel, for example by increasing the diameter of meningeal lymphatic vessel.
  • An example of a suitable FGF2 amino acid sequence in accordance with some embodiments is provided as Uniprot Accession No. P09038 (human FGF2) (SEQ ID NO: 4 -
  • Flow modulators e.g., FGF2 or VEGFR3 agonists such as VEGF-c
  • neurological therapeutic agents e.g., amyloid beta antibodies
  • Flow modulators e.g., FGF2 or VEGFR3 agonists such as VEGF-c
  • neurological therapeutic agents e.g., amyloid beta antibodies
  • Flow modulators can be administered to a subject using any of a number of suitable routes of administration, provided that the route of administration administers the flow modulator to the CNS (such as the meningeal space) of a subject. It is noted that many compounds do not readily cross the blood-brain barrier, and as such, some routes of administration such as intravenous will not necessarily deliver the flow modulator and/or neurological therapeutic agent to the CNS (unless the flow modulator can readily cross the blood-brain barrier).
  • administering to the CNS of a subject, as used herein (including variations of this root term), it is not necessarily required that a flow modulator and/or neurological therapeutic agent be administered directly to the CNS (such as meningeal space), but rather, this term encompasses administering a flow modulator and/or neurological therapeutic agent directly and/or indirectly to the CNS.
  • the flow modulator and/or neurological therapeutic agent so that it is in fluid communication with the CNS (e.g., meningeal space) of the subject in accordance with some embodiments herein (typically by administering the flow modulator and/or neurological therapeutic agent on the “brain” side of the blood-brain barrier), the flow modulator and/or neurological therapeutic agent will be administered to the meningeal space. Accordingly, in some embodiments, the flow modulator and/or neurological therapeutic agent is not administered systemically.
  • the flow modulator and/or neurological therapeutic agent is not administered systemically, but rather is administered to a fluid, tissue, or organ in fluid communication with the CNS (such as the meningeal space), and on the brain side of the blood-brain barrier.
  • the flow modulator and/or neurological therapeutic agent is not administered systemically, but rather is administered to the CNS.
  • the flow modulator and/or neurological therapeutic agent is administered to the CNS, but is not administered to any organ or tissue outside of the CNS.
  • the flow modulator and/or neurological therapeutic agent is not administered to the blood.
  • the flow modulator and/or neurological therapeutic agent is not administered to a tumor, or to the vasculature of a tumor.
  • a flow modulator and neurological therapeutic agent can be administered together (in a single composition), or separately (e.g., in separate compositions, which can be administered to the same location at the same or different times, or can be administered to different locations at the same or different times). Accordingly, in some embodiments, a flow modulator and neurological therapeutic agent can be administered together (in a single composition). In some embodiments, a flow modulator and neurological therapeutic agent are administered in separate compositions. In some embodiments, a flow modulator and neurological therapeutic agent are administered in separate compositions to different sites of administration on a subject at the same time.
  • a flow modulator and neurological therapeutic agent are administered in separate compositions to different sites of administration on a subject at different times (for example, the flow modulator can be administered prior to the neurological therapeutic agent, or the flow modulator can be administered after the neurological therapeutic agent).
  • a flow modulator and neurological therapeutic agent are administered in separate compositions to the same site of administration on a subject at different times (for example, the flow modulator can be administered prior to the neurological therapeutic agent, or the flow modulator can be administered after the neurological therapeutic agent).
  • Neurological diseases or disorders include, but are not limited to proteinopathies, for example tauopathies and/or amyloidoses such as AD (for example familial AD and/or sporadic AD).
  • Neurological diseases or disorders include, but are not limited to AD (for example familial AD and/or sporadic AD), dementia, age-related dementia, Parkinson’s disease (PD), cerebral edema, amyotrophic lateral sclerosis (ALS), Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections (PANDAS), meningitis, hemorrhagic stroke, autism spectrum disorder (ASD), brain tumor, and epilepsy.
  • AD familial AD and/or sporadic AD
  • dementia dementia
  • age-related dementia Parkinson’s disease
  • ALS amyotrophic lateral sclerosis
  • PANDAS Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections
  • ASD autism spectrum disorder
  • brain tumor and epilepsy.
  • the neurodegenerative disease is selected from the group consisting of: AD (such as familial AD and/or sporadic AD), PD, cerebral edema, ALS, PANDAS, meningitis, hemorrhagic stroke, ASD, brain tumor (such as glioblastoma), epilepsy, Down’s syndrome, hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D), Familial Danish/British dementia, dementia with Lewy bodies (DLB), Lewy body (LB) variant of AD, multiple system atrophy (MSA), familial encephalopathy with neuroserpin inclusion bodies (FENIB), frontotemporal dementia (FTD), Huntington’s disease (HD), Kennedy disease/spinobulbar muscular atrophy (SBMA), dentatorubropallidoluysian atrophy (DRPLA); spinocerebellar ataxia (SCA) type I, SAD (such as familial AD and/or sporadic AD
  • the neurological disease comprises, consists essentially of, or consists of a proteinopathy, for example AD (such as familial AD and/or sporadic AD), Down’s syndrome, HCHWA-D, Familial Danish/British dementia, PD, DLB, LB variant of AD, MSA, FENIB, ALS, FTD, HD, Kennedy disease/SBMA, DRPLA; SCA type I, SCA2, SCA3 (Machado-Joseph disease), SCA6, SCA7, SCA 17, CJD (such as familial CJD), Kura, GSS, FFI,
  • AD familial AD and/or sporadic AD
  • Down’s syndrome HCHWA-D
  • Familial Danish/British dementia PD
  • DLB DLB variant of AD
  • MSA FENIB
  • ALS Familial Danish/British dementia
  • FTD FTD
  • HD Kennedy disease/SBMA
  • DRPLA DRPLA
  • SCA type I S
  • the neurological disease or disorder is AD, dementia, or PD.
  • the neurological disease or disorder comprises, consists essentially of, or consists of a proteinopathy, for example a tauopathy and/or amyloidosis such as AD (for example familial AD and/or sporadic AD).
  • the neurological disease or disorder comprises, consists essentially of, or consists of AD (for example familial AD and/or sporadic AD).
  • biologically compatible form suitable for administration in vivo is meant a form of the flow modulator and/or neurological therapeutic agent to be administered in which any toxic effects are outweighed by the therapeutic effects of the flow modulator and/or neurological therapeutic agent.
  • subject is intended to include living organisms in which a neurological disease or disorder can be identified, e.g., mammals. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.
  • Administration of a flow modulator e.g., FGF2 or VEGFR3 agonist such as VEGF- c
  • neurological therapeutic agent e.g., amyloid beta antibody
  • a flow modulator e.g., FGF2 or VEGFR3 agonist such as VEGF- c
  • neurological therapeutic agent e.g., amyloid beta antibody
  • a flow modulator e.g., FGF2 or VEGFR3 agonist such as VEGF- c
  • neurological therapeutic agent e.g., amyloid beta antibody
  • a therapeutically active amount of a flow modulator and/or neurological therapeutic agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of peptide to elicit a desired response in the individual. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses can be administered daily or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • the flow modulator e.g.
  • FGF2 or VEGFR3 agonist such as VEGF-c) and/or neurological therapeutic agent (e.g., amyloid beta antibody) of the disclosure described herein can be administered in a convenient manner such as by transcranial administration, intrathecal administration, intraventricular administration, and/or intraparenchymal administration by contact with cerebral spinal fluid (CSF) of the subject, administration by pumping into CSF of the subject, administration by implantation into the skull or brain, administration by contacting a thinned skull or skull portion of the subject with the agent, injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, or rectal administration.
  • CSF cerebral spinal fluid
  • the flow modulator and/or neurological therapeutic agent are administered to the subject (or formulated for administration) by a route selected from the group consisting of: intrathecal administration, intraventricular administration, intraparenchymal administration, nasal administration, transcranial administration, contact with cerebral spinal fluid (CSF) of the subject, pumping into CSF of the subject, implantation into the skull or brain, contacting a thinned skull or skull portion of the subject with the neurological therapeutic agent, expression in the subject of a nucleic acid encoding the neurological therapeutic agent, intravenous infusion, or a combination of any of the listed routes.
  • a route selected from the group consisting of: intrathecal administration, intraventricular administration, intraparenchymal administration, nasal administration, transcranial administration, contact with cerebral spinal fluid (CSF) of the subject, pumping into CSF of the subject, implantation into the skull or brain, contacting a thinned skull or skull portion of the subject with the neurological therapeutic agent, expression in the subject of a nucleic acid encoding the neurological therapeutic agent, intrave
  • the flow modulator and/or neurological therapeutic agent can be administered to the subject (or formulated for administration) by a route selected from the group consisting of: intrathecal administration, intraventricular administration, intraparenchymal administration, nasal administration, transcranial administration, contact with cerebral spinal fluid (CSF) of the subject, pumping into CSF of the subject, implantation into the skull or brain, contacting a thinned skull or skull portion of the subject with the neurological therapeutic agent, expression in the subject of a nucleic acid encoding the neurological therapeutic agent, or a combination of any of the listed routes.
  • the flow modulator and the neurological therapeutic agent may be administered by the same route, or by different routes.
  • the neurological therapeutic agent is administered by intravenous infusion
  • the flow modulator is administered by any route of administration described herein.
  • the neurological therapeutic agent and the flow modulator are both administered by intravenous infusion.
  • the flow modulator and/or neurological therapeutic agent can be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound.
  • the neurological therapeutic agent alone, or with the flow modulator can be administered via intravenous infusion.
  • the intravenous infusion may be repeated, for example, once every 1 week, 2 weeks, 3 weeks, 4 weeks, month, or two months including ranges between any two of the listed values, for example, once every 1-2 weeks, 1-4 weeks, 1 week - 1 month, 2-4weeks, or 2 weeks - 1 month.
  • the neurological therapeutic agent may be a monoclonal antibody specific for amyloid beta, for example bapineuzumab, gantenerumab, aducanumab, solanezumab, and/or crenezumab.
  • the monoclonal antibody specific for amyloid beta such as bapineuzumab, gantenerumab, aducanumab, solanezumab, and/or crenezumab, is administered monthly via intravenous infusion.
  • the flow modulator e.g., FGF2 or VEGFR3 agonist such as
  • VEGF-c vascular endothelial growth factor-c
  • neurological therapeutic agent e.g., amyloid beta antibody
  • the flow modulator and/or neurological therapeutic agent can be provided in a nasal spray, or can be contacted directly with a nasal mucous membrane.
  • the flow modulator e.g., FGF2 or VEGFR3 agonist such as
  • VEGF-c vascular endothelial growth factor-c
  • neurological therapeutic agent e.g., amyloid beta antibody
  • the flow modulator and/or neurological therapeutic agent can be directly injected into CSF of a patient (for example into a ventricle of the brain).
  • Suitable apparatuses for injection can include a syringe, or a pump that is inserted or implanted in the subject and in fluid communication with CSF.
  • a composition comprising or consisting essentially of the flow modulator and/or neurological therapeutic agent, for example a slow-release gel, is implanted in a subject so that it is in fluid communication with CSF of the subject, and thus contacts the CSF.
  • the flow modulator e.g., FGF2 or VEGFR3 agonist such as
  • VEGF-c and/or neurological therapeutic agent is administered transcranially.
  • a composition comprising or consisting essentially of the flow modulator and/or neurological therapeutic agent such as a gel can be placed on an outer portion of the subject’s skull, and can pass through the subject’s skull.
  • the flow modulator and/or neurological therapeutic agent is contacted with a thinned portion of the subject’s skull to facilitate transcranial delivery.
  • the flow modulator e.g., FGF2 or VEGFR3 agonist such as
  • VEGF-c and/or neurological therapeutic agent e.g., amyloid beta antibody
  • a vector comprising or consisting essentially of the nucleic acid for example a viral vector such as a retroviral vector, lentiviral vector, or adenoviral vector, or adeno-associated viral vector (AAV) can be administered to a subject as described herein, for example via injection or inhalation.
  • a viral vector such as a retroviral vector, lentiviral vector, or adenoviral vector, or adeno-associated viral vector (AAV)
  • AAV adeno-associated viral vector
  • the nucleic acid is administered as an mRNA as described herein, for example as a chemically modified messenger RNA (mRNA).
  • expression of the nucleic acid is induced in the subject, for example via a drug or optical regulator of transcription.
  • the flow modulator e.g. the VEGFR3 antagonist such as
  • VEGF-c or FGF2 vascular endothelial growth factor-2
  • neurological therapeutic agent e.g. , amyloid beta antibody
  • administered “selectively” and variations of the root term indicate that the flow modulator is administered preferentially to the indicated target (e.g. meningeal space) compared to other tissues or organs on the same side of the blood brain barrier.
  • target e.g. meningeal space
  • direct injection to meningeal spaces of the brain would represent “selective” administration, whereas administration to CSF in general via a spinal injection would not.
  • the flow modulator and/or neurological therapeutic agent is administered selectively to the meningeal space, and not to portions of the CNS outside of the meningeal space, nor to any tissues or organs outside of the CNS. In some embodiments, the flow modulator is administered selectively to the CNS, and not to tissue or organs outside of the CNS such as the peripheral nervous system, muscles, the gastrointestinal system, musculature, or vasculature.
  • a flow modulator e.g., FGF2 or VEGFR3 agonist such as VEGF-c
  • neurological therapeutic agent e.g., amyloid beta antibody
  • the flow modulator and/or neurological therapeutic agent as described herein can be administered via a route of administration as described herein hourly, daily, every other day, every three days, every four days, every five days, every six days, weekly, biweekly, monthly, bimonthly, and the like.
  • the flow modulator and/or neurological therapeutic agent is administered in a single administration, but not in any additional administrations.
  • Some embodiments include methods of making a composition or medicament comprising or consisting essentially of a flow modulator (e.g., FGF2 or VEGFR3 agonist such as VEGF-c) and/or neurological therapeutic agent (e.g., amyloid beta antibody) as described herein suitable for administration according to a route of administration as described herein.
  • a composition comprising or consisting essentially of a VEGFR3 agonist (such as VEGF-c) and an amyloid beta antibody is prepared for nasal administration, administration to the CSF, or transcranial administration.
  • a composition comprising or consisting essentially of a VEGFR3 antagonist (such as VEGF-c) and amyloid beta antibody is prepared for nasal administration, administration by contacting with CSF, or transcranial administration.
  • a pharmaceutical composition comprising a flow modulator (e.g. the VEGFR3 antagonist or FGF2) and/or neurological therapeutic agent (e.g, amyloid beta antibody) to the subject, depending upon the type of disease to be treated or the site of the disease.
  • This composition can also be administered via other conventional routes, e.g., administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
  • parenteral includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrastemal, intrathecal, intraventricular, intraparenchymal, intralesional, and intracranial injection or infusion techniques.
  • it can be administered to the subject via injectable depot routes of administration such as using 1-, 3-, or 6-month depot injectable or biodegradable materials and methods.
  • the pharmaceutical composition is administered intraocularly or intravitreally.
  • the pharmaceutical composition comprising the flow modulator
  • VEGFR3 antagonist or FGF2 e.g. the VEGFR3 antagonist or FGF2
  • neurological therapeutic agent e.g., amyloid beta antibody
  • intrathecally, intraventricularly, and/or intraparenchymally e.g., via an injection into the spinal canal, or into the subarachnoid space.
  • the flow modulator e.g., FGF2 or VEGFR3 agonist such as
  • VEGF-c and/or neurological therapeutic agent e.g., amyloid beta antibody
  • the flow modulator and/or neurological therapeutic agent is administered intraventricularly, i.e., into the lateral ventricle of the brain.
  • the flow modulator and/or neurological therapeutic agent is administered intraparenchymally, i.e., into the brain parenchyma.
  • the flow modulator and/or neurological therapeutic agent is administered and delivered through an implanted catheter connected to a pump, which contains a reservoir of the composition and controls the rate of delivery.
  • the flow modulator and/or neurological therapeutic agent is released into the cerebrospinal fluid (CSF) of the cistema magna.
  • CSF cerebrospinal fluid
  • the catheter can either be introduced between the first and second cervical vertebrae (C1-C2 interspace) or into the intracranial ventricles.
  • an Ommaya reservoir (consisting of a catheter in one lateral ventricle attached to a reservoir implanted under the scalp) is used as an intraventricular catheter system for the administration and delivery of the flow modulator and/or neurological therapeutic agent into the cerebrospinal fluid.
  • the flow modulator e.g., FGF2 or VEGFR3 agonist such as
  • VEGF-c and/or neurological therapeutic agent (e.g., amyloid beta antibody) is administered at the site of an amyloid beta plaque.
  • neurological therapeutic agent e.g., amyloid beta antibody
  • Injectable compositions may contain various carriers such as vegetable oils, dimethylactamide, dimethyformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, and the like).
  • various carriers such as vegetable oils, dimethylactamide, dimethyformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, and the like).
  • water soluble compositions comprising flow modulator (e.g., FGF2 or VEGFR3 agonist such as VEGF-c) and/or neurological therapeutic agent (e.g., amyloid beta antibody) can be administered by the drip method, whereby a pharmaceutical formulation containing the flow modulator and/or neurological therapeutic agent and a physiologically acceptable excipient is infused.
  • Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer’s solution or other suitable excipients.
  • Intramuscular preparations e.g., a sterile formulation of a suitable soluble salt form of the flow modulator and/or neurological therapeutic agent
  • a pharmaceutical excipient such as Water-for-Injection, 0.9% saline, or 5% glucose solution.
  • the composition comprises VEGFR3 agonist and/or FGF2.
  • a flow modulator e.g., FGF2 or VEGFR3 agonist such as
  • VEGF-c and/or neurological therapeutic agent is administered via site- specific or targeted local delivery techniques.
  • site-specific or targeted local delivery techniques include various implantable depot sources of the flow modulator or local delivery catheters, such as infusion catheters, an indwelling catheter, or a needle catheter, synthetic grafts, adventitial wraps, shunts and stents or other implantable devices, site specific carriers, direct injection, or direct application. See, e.g., PCT Publication No. WO 00/53211 and U.S. Pat. No. 5,981,568.
  • the flow modulator e.g., FGF2 or VEGFR3 agonist such as VEGF-c
  • neurological therapeutic agent e.g., amyloid beta antibody
  • Targeted delivery of therapeutic compositions comprising a flow modulator and/or neurological therapeutic agent as described herein
  • an antisense polynucleotide, expression vector (viral or non-viral), or subgenomic polynucleotides, or mRNA is also contemplated within the disclosure.
  • Receptor-mediated DNA delivery techniques are described in, for example, Findeis et al, Trends Biotechnol. (1993) 11:202; Chiou et al, Gene Therapeutics: Methods and Applications of Direct Gene Transfer (J. A. Wolff, ed.) (1994); Wu et al, J. Biol. Chem. (1988) 263:621; Wu et al, J. Biol. Chem. (1994) 269:542; Zenke etal, Proc. Natl. Acad. Sci. USA (1990) 87:3655; Wu etal, J. Biol. Chem. (1991) 266:338.
  • compositions containing a polynucleotide are administered in a range of about 100 ng to about 200 mg of DNA for local administration in a gene therapy protocol.
  • ranges of about 500 ng to about 50 mg, about 1 pg to about 2 mg, about 5 pg to about 500 pg, and about 20 pg to about 100 pg of DNA or more can also be used during a gene therapy protocol.
  • DNA is administered at a concentration of about 100 ng/ml to about 200 mg/ml.
  • the flow modulator e.g. , FGF2 and/or VEGFR3 agonists such as VEGF-c, described herein
  • neurological therapeutic agent e.g., amyloid beta antibody
  • the gene delivery vehicle can be of viral or non-viral origin (see generally, Jolly, Cancer Gene Therapy (1994) 1:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy (1995) 1: 185; and Kaplitt, Nature Genetics (1994) 6: 148).
  • Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters and/or enhancers. Expression of the coding sequence can be either constitutive or regulated.
  • Viral-based vectors for delivery of a desired polynucleotide (for example, encoding a flow modulator and/or neurological therapeutic agent as described herein) and expression in a desired cell are well known in the art.
  • Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S. Pat. Nos. 5,219,740 and 4,777,127; GB Patent No.
  • alphavirus-based vectors e.g., Sindbis virus vectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitis vims (ATCC VR-923; ATCC VR- 1250; ATCC VR 1249; ATCC VR-532)
  • AAV adeno-associated vims
  • AAV vectors are particularly suitable for delivery of a payload into the central nervous system.
  • Over 100 AAV serotypes have been identified that differ in the binding capacity of capsid proteins to specific cell surface receptors that can transduce different cell types and brain regions in the CNS.
  • Non-limiting examples of AAV serotypes include AAV1, AAV2/1, AAVDJ, AAV8, AAVDJ8, AAV9, and AAVDJ9.
  • Promoters to drive expression in the brain can be constitutive, such as beta-actin, phosphoglycerate kinase 1 or CMV promoters, or tissue specific.
  • tissue specific promoters which can be used to drive expression in brain tissues include the synapsin, Glial fibrillary acidic protein (GFAP), glutamic acid decarboxylase (GAD67), homeobox Dlx5/6, glutamate receptor 1 (GluRl), and preprotachykinin 1 (Tael), and Musashil promoters. These promoters show diversity of transcriptional activity and cell-type specificity of expression. Accordingly, in some embodiments, a promoter is selected based on the desired expression in a cell type, tissue or brain region.
  • the expression of the nucleic acid of the interest is under control of a brain tissue specific promoter, including but not limited to, synapsin, GFAP, GAD67, homeobox Dlx5/6, GluRl, and Tael, and Musashil promoters or others known in the art.
  • a brain tissue specific promoter including but not limited to, synapsin, GFAP, GAD67, homeobox Dlx5/6, GluRl, and Tael, and Musashil promoters or others known in the art.
  • hTERT promoter has been used to drive cancer-specific expression in a number different types of cancer tissues.
  • Alpha-fetoprotein (AFP) and erb2 promoters have been used to target hepatic cancer and breast cancer, respectively.
  • AFP carcinoembryonic antigen
  • COX-2 cyclooxygenase -2
  • hTERT hTERT
  • Urokinase-type plasminogen activator receptor Several promoters, including carcinoembryonic antigen (CEA), cyclooxygenase -2 (COX-2), hTERT, and Urokinase-type plasminogen activator receptor (uPAR) have been used to direct suicide genes into colorectal carcinoma cells (Rama et al, Disease Markers (2015).
  • a promoter active under hypoxic conditions can be used tumor specific expression in the tumor microenvironment, such as the HIF-1 promoter.
  • a hypoxia- inducible promoter e.g., p53 for induction of apoptosis, HSV thymidine kinase, bacterial nitroreductase, VEGF receptor 1-Ig, CD40 ligand, and IF-4 (see, e.g., Guo, Virus Adaptation and Treatment 2011:371-82 The impact of hypoxia on oncolytic virotherapy).
  • the expression of the nucleic acid of the interest is under control of a tumor specific promoter, including but not limited to, hTERT, AFP, erb2 CEA, COX-2, and uPAR promoters, or a hypoxia inducible promoter, including but not limited to HIF 1.
  • a tumor specific promoter including but not limited to, hTERT, AFP, erb2 CEA, COX-2, and uPAR promoters, or a hypoxia inducible promoter, including but not limited to HIF 1.
  • non-viral delivery vehicles and methods are employed, including, but not limited to, polycationic condensed DNA linked or unlinked to killed adenovirus alone (see, e.g., Curiel, Hum. Gene Ther. (1992) 3:147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989) 264:16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S. Pat. No. 5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO 95/30763; and WO 97/42338) and nucleic charge neutralization or fusion with cell membranes.
  • polycationic condensed DNA linked or unlinked to killed adenovirus alone see, e.g., Curiel, Hum. Gene Ther. (1992) 3:147
  • ligand-linked DNA see, e.g., Wu, J. Biol. Chem. (1989) 264
  • Naked DNA can also be employed.
  • Exemplary naked DNA introduction methods are described in PCT Publication No. WO 90/11092 and U.S. Pat. No. 5,580,859.
  • Liposomes that can act as gene delivery vehicles are described in U.S. Pat. No. 5,422,120; PCT Publication Nos. WO 95/13796; WO 94/23697; WO 91/14445; and EP Patent No. 0524968. Additional approaches are described in Philip, Mol. Cell. Biol. (1994) 14:2411, and in Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.
  • the flow modulator e.g., FGF2 or VEGFR3 agonist such as
  • VEGF-c and/or neurological therapeutic agent is administered as an mRNA (e.g., a mRNA encoding the VEGFR3 agonist).
  • mRNA chemically modified messenger RNA
  • Modified mRNA evades recognition by the innate immune system and is less immunostimulating than dsDNA or regular mRNA.
  • cytoplasmic delivery of mRNA circumvents the nuclear envelope, which can result in a higher expression level.
  • Exemplary mRNA introduction methods are described in Rhoads et al. (Methods in Molecular Biology, vol. 1428, DOI 10.1007/978-1-4939-3625-01), and references therein.
  • nucleic acids e.g., comprising or encoding a flow modulator and/or neurological therapeutic agent
  • a cell naked i. e. , free from complexing agents, for example, lipid agents and polymer agents, etc.
  • naked mRNA is delivered by injection (intradermal, intrathecal, intraventricular, intraparenchymal, etc).
  • the flow modulator and/or neurological therapeutic agent nucleic acids or polypeptides of compositions and methods of some embodiments may be formulated according to methods known in the art, and the formulations may further include, but are not limited to, cell penetration agents, a pharmaceutically acceptable carrier, delivery agents, a bioerodible or biocompatible polymers, solvents, and sustained-release delivery depots.
  • the nucleic acid comprising or encoding the flow modulator and/or neurological therapeutic agent comprises, consists essentially of, or consists of modified nucleic acids or nucleobases.
  • Example flow modulators that can be encoded include VEGFR3 agonist or antagonist, or FGF2.
  • Example neurological therapeutic agents include amyloid beta antibodies.
  • the nucleic acid encoding the flow modulator and/or neurological therapeutic agent comprises, consists essentially of, or consists of an antisense oligonucleotide (ASO).
  • ASO antisense oligonucleotide
  • the ASO can hybridize to a complementary mRNA and mediate silencing of expression from the mRNA, such as by blocking ribosome binding to the mRNA and/or recruiting RNase H to mediate degradation of the mRNA.
  • the nucleic acid molecule can be a mimetic, can include a modified sugar backbone, one or more modified intemucleoside linkages (e.g., one or more phosphorothioate and/or heteroatom intemucleoside linkages), one or more modified bases, and the like.
  • the nucleic acid has a morpholino backbone structure.
  • the nucleic acid has one or more locked nucleic acids (LNAs).
  • the nucleic acid has base modifications. Base modifications include synthetic and natural nucleobases.
  • Suitable base modifications include pyridin- 4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio- pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl- pseudouridine, 5-propynyl-uridine, 1-propynyl -pseudouridine, 5-taurinomethyhiridine, 1- taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, l-taurinomethyl-4-thio-uridine, 5- methyl -uridine, 1-methylpseudouridine, 4-thio-l -methyl -pseudouridine, 2-thio-l -methyl- pseudouridine, 1 -methyl- 1 -deaza-pseudouridine, 2-thio- 1 -methyl- 1 -
  • the flow modulator and/or neurological therapeutic agent can comprise naturally occurring and/or artificial nucleic acid, for example a mimetic, one or more modified intemucleoside linkages, and/or one or more modified bases, such as base modifications as described herein.
  • the nucleic acid (e.g., of the flow modulator and/or neurological therapeutic agent) is an mRNA which has at least one modification in one of the bases A,G, U and/or C.
  • the mRNA has at least one 5' terminal cap is selected from the group consisting of CapO, Cap 1, ARCA, inosine, N1 -methyl -guanosine, 2'fluoro-guanosine, 7- deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine and 2-azido-guanosine.
  • the mRNA has a polyA or a polyT tail, for example, of 100-200 nucleotides.
  • the UTR(s) are not derived from the native untranslated region corresponding to the polypeptide of interest, e.g., are beta-globin UTRs.
  • the sequence composition of the mRNA is altered by incorporating the most GC-rich codon for each amino acid. For example, expression of proteins from synthetic mRNA can be diminished if the mRNA contains rare codons or rate-limiting regulatory sequences.
  • the nucleic acids of the disclosure are codon optimized, e.g., to optimize translation and reduce immunogenicity.
  • Codon optimization tools known in the art employ algorithms for codon optimization, many of which take codon usage tables, codon adaptability, mRNA structure, and various cis-elements in transcription and translation into consideration.
  • a non-limiting example of a useful platform is ptimumGeneTM algorithm from GenScript.
  • mRNA is produced according to methods known in the art (see e.g., Rhoads (ed.), Synthetic mRNA: Production, Introduction Into Cells, and Physiological Consequences, Methods in Molecular Biology, vol. 1428, DOI 10.1007/978-l-4939-3625-0_l).
  • mRNA is synthesized in vitro, e.g., using T7 polymerase- mediated transcription from a linearized DNA template containing an open reading frame, flanking 5' and 3' untranslated regions and a poly-A tail.
  • a Cap structure such as a Capl structure, can be enzymatically added to the 5' end to produce the final mRNA.
  • the nucleobases bases are modified.
  • uridine is completely substituted with Nl- methylpseudouridine to reduce potential immunostimulatory activity and to improve protein expression relative to unmodified mRNA.
  • one or more of the modifications described supra can be used.
  • Suitable buffer solutions are known in the art.
  • a non-limiting example of a suitable buffer is a solution containing 2.94 mg/mL sodium citrate dihydrate at pH 6.5 and 7.6 mg/mL sodium chloride (Gan et al, Nature Communications, volume 10, Article number: 871 (2019)).
  • the nucleic acid (e.g., comprising or encoding a flow modulator and/or neurological therapeutic agent) includes a conjugate moiety (e.g., one that enhances the activity, cellular distribution or cellular uptake of the oligonucleotide).
  • conjugate moiety e.g., one that enhances the activity, cellular distribution or cellular uptake of the oligonucleotide.
  • conjugate groups can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups.
  • Conjugate groups include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers.
  • Suitable conjugate groups include, but are not limited to, cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.
  • Groups that enhance the pharmacodynamic properties include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid.
  • Groups that enhance the pharmacokinetic properties include groups that improve uptake, distribution, metabolism or excretion of a nucleic acid.
  • the particular dosage regimen i.e., dose, timing and repetition, used in the methods of some embodiments herein depend on the particular subject and that subject's medical history.
  • more than one flow modulator and/or neurological therapeutic agent may be administered to a subject in need of the treatment.
  • the flow modulator e.g., FGF2 and/or VEGFR3 agonist such as VEGF-c
  • neurological therapeutic agent e.g., amyloid beta antibody
  • the flow modulator and/or neurological therapeutic agent is administered.
  • Treatment efficacy for a target neurological disease/disorder for example a proteinopathy (e.g. , a tauopathy and/or amyloidosis such as AD), for example in the head, skull, meninges, central nervous system, and/or brain as described herein can be assessed by methods well- known in the art.
  • the target neurological disease/disorder can comprise amyloid beta plaques.
  • a flow modulator e.g. , FGF2 or VEGFR3 agonist such as VEGF-c
  • neurological therapeutic agent e.g., amyloid beta antibody
  • a pharmaceutically acceptable carrier excipient
  • the carrier must be compatible with the active ingredient of the composition (and preferably, capable of stabilizing the active ingredient) and not deleterious to the subject to be treated.
  • compositions including buffers, which are well known in the art. See, e.g., Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover.
  • compositions to be used in the present methods can comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions.
  • pharmaceutically acceptable carriers excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used, and may comprise buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine,
  • the pharmaceutical composition described herein comprises liposomes containing the flow modulator (e.g., FGF2 or VEGFR3 agonist such as VEGF-c) and/or neurological therapeutic agent (e.g., amyloid beta antibody) (or the encoding nucleic acids) which can be prepared by methods known in the art, such as described in Epstein, et al. , Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang, etal., Proc. Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat.
  • the flow modulator e.g., FGF2 or VEGFR3 agonist such as VEGF-c
  • neurological therapeutic agent e.g., amyloid beta antibody
  • Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE).
  • PEG-PE PEG-derivatized phosphatidylethanolamine
  • the liposomes can be extruded through fdters of defined pore size to yield liposomes with the desired diameter. Without being limited by theory, it is contemplated that minimizing lipid diameter can inhibit or avoid interaction between the liposome and circulating proteins, thus prolonging the circulation time of the liposome.
  • Lipsomes for mRNA delivery reviewed, for example, in Reichmuth et al, Ther. Deliv. 7: 319-334 (2016), which is incorporated by reference in its entirety herein.
  • the liposome has a diameter smaller than the interior diameter of a meningeal lymphatic vessel, so that the liposome may travel through the meningeal lymphatics.
  • the liposome has a dimeter of less than 150 nm, for example, less than 140 nm, 130 nm, 120 nm, 110 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm, or 10 nm, including ranges between any two of the listed values.
  • a target cell can endocytose of a liposome comprising mRNA, and following release of the mRNA from the lipsome, the mRNA can be available in the cytosol of the target cell.
  • the inclusion of an amine group at or near the surface of the liposome can maintain a neutral or mildly cationic surface charge at physiological pH, so as to minimize non-specific protein interactions can facilitate release of the mRNA in the cytosol.
  • the liposomes are administered to a subject in vivo according to a route of administration as described herein, for example parenteral or intranasal.
  • the flow modulator e.g., FGF2 or VEGFR3 agonist such as
  • VEGF-c and/or neurological therapeutic agent e.g., amyloid beta antibody
  • mRNA is contained in 3 -[N-(N',N'-dimethylaminoethane) carbamoyl](DC-Cholesterol)/dioleoylphosphatidylethanolamine (DOPE) liposomes.
  • the VEGFR3 agonist is encapsulated with DOTAP.
  • the VEGFR3 agonist is encapsulated in a cationic lipid preparation.
  • RNA can also be protected against degradation by complexing with the poly cationic protein protamine.
  • VEGFR3 agonist mRNA is complexed with protamine, for example, according to the curevac RNActive® platform.
  • protamine for example, according to the curevac RNActive® platform.
  • complexing with protamine is contemplated to inhibit or limit immunogenicity of the composition comprising the mRNA.
  • Excipients suitable for flow modulators e.g. , FGF2 or VEGFR3 agonist such as
  • VEGF-c and/or neurological therapeutic agents can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, carbohydrates, cells loaded with nucleic acids or polypeptides of the disclosure, hyaluronidase, nanoparticle mimics and combinations thereof.
  • nucleic acids can be delivered using a Gene Gun.
  • nucleic acids e.g., DNA, mRNA, siRNA, etc.
  • lipidoids may be formulated in lipidoids.
  • Non-limiting examples of such lipidoids contain amino-alkyl -acrylate and - acrylamide materials and are known in the art (see e.g., Love et al, PNAS May 25, 2010 107 (21) 9915).
  • C16-96, C14-110, and C12-200 are other examples of lipidoids, which can be prepared, complexed with nucleic acid, e.g., siRNA or mRNA, and delivered according to Love et al, PNAS May 25, 2010 107 (21) 9915.
  • the nucleic acid of the disclosure is administered in stable nucleic acid lipid particle (SNALP) formulations.
  • SNALP stable nucleic acid lipid particle
  • nucleic acids or polypeptides described herein may be formulated in lipid nanoparticles.
  • the formulation may be influenced by parameters including, but not limited to, the selection of the cationic lipid component, the degree of cationic lipid saturation, the nature of the PEGylation, ratio of all components and biophysical parameters such as size (Semple et al Nature Biotech. 201028:172-176).
  • a non-limiting example of a cationic lipid that is suitable for formulation of nucleic acids e.g., mRNA, l,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA).
  • This cationic lipid can be synthesized and used as a main component of lipid nanoparticles.
  • An ethanol dilution process is used to produce small uniform lipid particles with a high RNA encapsulation efficiency (Geall et al, PNAS September 4, 2012 109 (36) 14604-14609).
  • nucleic acids or polypeptides described herein may be formulated in exosomes, microvesicles, and/or extracellular vesicles.
  • the exosomes, microvesicles, and/or extracellular vesicles may be loaded with at least one VEGFR3 agonist or VEGFR3 antagonist and delivered to cells or tissues.
  • Exosomes which can function as nucleic acid delivery vehicles are known in the art and are for example described in US 9629929.
  • the flow modulator e.g. , FGF2 or VEGFR3 agonist such as VEGF-c
  • neurological therapeutic agent e.g., amyloid beta antibody
  • the encoding nucleic acid(s) may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • the pharmaceutical composition described herein can be formulated in sustained-release format.
  • sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide or nucleic acid of the disclosure, which matrices are in the form of shaped articles, e.g. fdms, or microcapsules.
  • sustained-release matrices include polyesters, hydrogels (for example, poly(2- hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactides (U.S. Pat. No.
  • copolymers of L-glutamic acid and 7 ethyl-L-glutamate copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid).
  • LUPRON DEPOTTM injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate
  • sucrose acetate isobutyrate sucrose acetate isobutyrate
  • poly-D-(-)-3-hydroxybutyric acid poly-D-(-)-3-hydroxybutyric acid
  • the compositions described herein may include at least one polymer such as, but not limited to, polyethenes, polyethylene glycol (PEG), poly(l- lysine)(PLL), PEG grafted to PLL, cationic lipopolymer, biodegradable cationic lipopolymer, polyethylenimine (PEI), cross-linked branched poly(alkylene imines), a polyamine derivative, a modified poloxamer, a biodegradable polymer, elastic biodegradable polymer, biodegradable block copolymer, biodegradable random copolymer, biodegradable polyester copolymer, biodegradable polyester block copolymer, biodegradable polyester block random copolymer, multiblock copolymers, linear biodegradable copolymer, poly[a-(4-aminobutyl)-L-glycolic acid) (PAGA), biodegradable cross- linked cationic multi-block copolymers, PAGA), biodegrad
  • a self-assembling polyplex nanomicelle composed of a polyethylene glycol- polyamino acid block copolymer was used to administer luciferase-expressing mRNA with nucleoside modification into the CNS by intrathecal injection into the cistema magna of mice (Ushida et al. ,
  • the nucleic acid, e.g., mRNA, of the disclosure is delivered intrathecally in a polyplex nanomicelle composed of a polyethylene glycol- polyamino acid block copolymer.
  • compositions of some embodiments herein are to be used for in vivo administration, and are sterile. Sterilization can be readily accomplished by, for example, filtration through sterile filtration membranes.
  • Therapeutic flow modulator and/or neurological therapeutic agent compositions are generally placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • compositions described herein can be in unit dosage forms such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration, or administration by inhalation or insufflation.
  • the principal active ingredient can be mixed with a pharmaceutical carrier, e.g., conventional tableting ingredients such as com starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g., water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or a non-toxic pharmaceutically acceptable salt thereof.
  • a pharmaceutical carrier e.g., conventional tableting ingredients such as com starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g., water
  • preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules.
  • This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from 0.1 to about 500 mg of the active ingredient of the present invention.
  • the tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action.
  • the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former.
  • Suitable surface -active agents include, in particular, non-ionic agents, such as polyoxyethylenesorbitans (e.g., TweenTM 20, 40, 60, 80 or 85) and other sorbitans (e.g., SpanTM 20,
  • compositions with a surface-active agent will conveniently comprise between 0.05 and 5% surface -active agent, and can be between 0.1 and 2.5%. It will be appreciated that other ingredients may be added, for example mannitol or other pharmaceutically acceptable vehicles, if necessary.
  • Suitable emulsions may be prepared using commercially available fat emulsions, such as IntralipidTM, LiposynTM, InfonutrolTM, LipofundinTM and LipiphysanTM.
  • the active ingredient may be either dissolved in a pre-mixed emulsion composition or alternatively it may be dissolved in an oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, com oil or almond oil) and an emulsion formed upon mixing with a phospholipid (e.g. egg phospholipids, soybean phospholipids or soybean lecithin) and water.
  • an oil e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, com oil or almond oil
  • a phospholipid e.g. egg phospholipids, soybean phospholipids or soybean lecithin
  • Suitable emulsions will typically contain up to 20% oil, for example, between 5 and 20%.
  • the emulsion compositions can be those prepared by mixing an VEGFR3 agonist or
  • VEGFR3 antagonist with IntralipidTM or the components thereof (soybean oil, egg phospholipids, glycerol and water).
  • compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders.
  • the liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as set out above.
  • the compositions are administered by the oral or nasal respiratory route for local or systemic effect.
  • compositions in preferably sterile pharmaceutically acceptable solvents may be nebulized by use of gases. Nebulized solutions may be breathed directly from the nebulizing device or the nebulizing device may be attached to a face mask, tent or intermittent positive pressure breathing machine. Solution, suspension or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner.
  • the flow modulator e.g. , FGF2 or VEGFR3 agonist such as VEGF-c
  • neurological therapeutic agent e.g., amyloid beta antibody
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
  • compositions of a flow modulator e.g., FGF2 or VEGFR3 agonist such as VEGF-c
  • neurological therapeutic agent e.g., amyloid beta antibody
  • a flow modulator e.g., FGF2 or VEGFR3 agonist such as VEGF-c
  • neurological therapeutic agent e.g., amyloid beta antibody
  • sterile aqueous solutions where water soluble or dispersions
  • sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • the composition will in some embodiments be sterile and must be fluid to the extent that the composition has easy syringeability (such as the composition easily passing from a container through an injection needle into a syringe prior to injection) and injectability (such as the composition easily passes from a syringe through an injection needle into an administration site of the subject)), See, e.g., Cilurzo et al. AAPS PharmSciTech. 12: 604-609 (2011) for a review of syringeability and injectability). It will in some embodiments be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating a flow modulator (e.g. ,
  • FGF2 or VEGFR3 agonist such as VEGF-c) and/or neurological therapeutic agent (e.g., amyloid beta antibody) of the disclosure in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by fdtered sterilization.
  • VEGF-c vascular endothelial growth factor
  • neurological therapeutic agent e.g., amyloid beta antibody
  • dispersions are prepared by incorporating the active flow modulator and/or neurological therapeutic agent compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions and, in some embodiments methods of preparation are vacuum drying and freeze- drying which yields a powder of the flow modulator and/or neurological therapeutic agent plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the flow modulator and/or neurological therapeutic agent can be orally administered, for example, with an inert diluent or an assimilable edible carrier.
  • pharmaceutically acceptable carrier has its ordinary meaning as understood in the art in view of the specification, and includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art.
  • Dosage unit form has its ordinary meaning as understood in the art in view of the specification, and includes physically discrete units suited as unitary dosages for the mammalian subjects (such as humans) to be treated; each unit containing a predetermined quantity of active compound (e.g., flow modulator and/or neurological therapeutic agent) calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • active compound e.g., flow modulator and/or neurological therapeutic agent
  • a flow modulator and/or neurological therapeutic agent of the disclosure is an antibody.
  • a therapeutically effective amount of antibody ranges from about 0.001 to 30 mg kg body weight, in some embodiments about 0.01 to 25 mg kg body weight, in some embodiments about 0.1 to 20 mg kg body weight, and in some embodiments about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg kg, 4 to 7 mg/kg, or 5 to 6 mg kg, or a range defined by any two of the preceding values, body weight.
  • an effective dosage ranges from about 0.001 to 30 mg kg body weight, in some embodiments about 0.01 to 25 mg kg body weight, in some embodiments about 0.1 to 20 mg kg body weight, and in some embodiments about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg kg, 4 to 7 mg/kg, or 5 to 6 mg kg, or a range defined by any two of the preceding values, body weight.
  • an effective dosage ranges from about 0.001 to 30 mg kg body weight, in some embodiments about 0.01 to 25 mg kg body weight, in some embodiments about 0.1 to 20 mg
  • treatment of a subject with a therapeutically effective amount of a flow modulator e.g. , FGF2 or VEGFR3 agonist such as VEGF-c
  • a neurological therapeutic agent such as an amyloid beta antibody
  • a flow modulator e.g. , FGF2 or VEGFR3 agonist such as VEGF-c
  • neurological therapeutic agent such as an amyloid beta antibody
  • a subject is treated with antibody (such as a neurological therapeutic agent comprising, consisting essentially of, or consisting of an amyloid beta antibody) in the range of between about 0.1 to 20 mg kg body weight, one time per week for between about 1 to 10 weeks, in some embodiments between 2 to 8 weeks, in some embodiments between about 3 to 7 weeks, and in some embodiments for about 4, 5, or 6 weeks, or a range defined by any two of the preceding values.
  • antibody such as a neurological therapeutic agent comprising, consisting essentially of, or consisting of an amyloid beta antibody
  • the effective dosage of antibody (e.g., amyloid beta antibody) used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result from the results of diagnostic assays.
  • an antibody of the disclosure can also be administered in combination therapy with, e.g., chemotherapeutic agents, hormones, antiangiogens, radiolabeled, compounds, or with surgery, cryotherapy, and/or radiotherapy.
  • An antibody of the disclosure e.g., amyloid beta antibody
  • additional forms of therapy such as one or more conventional therapies, which may include, for example, an antibody, peptide, a fusion protein and/or small molecule
  • additional therapies such as one or more conventional therapies, which may include, for example, an antibody, peptide, a fusion protein and/or small molecule
  • the antibody can be administered with a therapeutically effective dose of chemotherapeutic agent.
  • the antibody can be administered in conjunction with chemotherapy to enhance the activity and efficacy of the chemotherapeutic agent.
  • the Physicians' Desk Reference discloses dosages of chemotherapeutic agents that have been used in the treatment of various neurological disease and disorders.
  • the dosing regimen and dosages of these aforementioned chemotherapeutic drugs that are therapeutically effective will depend on the particular neurological disease or disorder, being treated, the extent of the disease and other factors familiar to the physician of skill in the art and can be determined by the physician.
  • the flow modulators e.g. , FGF2 or VEGFR3 agonists such as VEGF-c
  • neurological therapeutic agents e.g., amyloid beta antibodies
  • nanoparticle-based composition and delivery methods well known to the skilled artisan.
  • nanoparticle-based delivery for improved nucleic acid (e.g., small RNAs) therapeutics are well known in the art (Expert Opinion on Biological Therapy 7: 1811- 1822).
  • a flow modulator e.g. , FGF2 or VEGFR3 agonist such as VEGF-c
  • neurological therapeutic agent e.g., amyloid beta antibody
  • an appropriate carrier e.g., VEGF2 or VEGFR3 agonist such as VEGF-c
  • pharmaceutically acceptable diluents include saline and aqueous buffer solutions.
  • Adjuvant is used in its broadest sense and includes any immune stimulating compound such as interferon.
  • Adjuvants contemplated herein include resorcinols, non- ionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether.
  • Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEEP) and trasylol.
  • Liposomes include water-in-oil-in-water emulsions as well as conventional liposomes (Sterna etal. (1984) J. Neuroimmunol. 7:27).
  • the present disclosure provides therapy or treatment of neurodegenerative diseases using neurological therapeutic agents in combination with flow modulators.
  • the neurological therapeutic agent e.g., an amyloid beta antibody
  • the flow modulator e.g., VEGFR3 agonist and/or FGF2
  • flow modulators increase fluid flow in the central nervous system (e.g., brain) and as a result improve the delivery of the neurological therapeutic agents and/or drainage of the lymphatic vessel within the central nervous system (e.g., brain).
  • improved drainage allows the removal of pathological aggregates targeted by the therapeutic agent.
  • a treatment combining a flow modulator described herein with a therapeutic agent described herein may improve the extent of the desired effect on the pathology of the neurological disease, e.g., reduction of pathological protein aggregates, reduction of inflammation at the site of aggregation, etc., as compared to a treatment with the therapeutic agent alone or as compared to a treatment with the flow modulator alone.
  • a “neurological therapeutic agent” refers to an agent that treats, prevents, inhibits, ameliorates, or reduces the symptoms of one or more neurological diseases, for example proteinopathies as described herein (e.g., tauopathies and/or amyloidoses such as AD).
  • the neurological therapeutic agent is selected from a group consisting of a small molecule, an oligopeptide, a polypeptide, an antibody, a nucleic acid, a recombinant virus, a vaccine, and a cell.
  • the neurological therapeutic agent may target a peptide or a protein that is involved in the pathogenesis of a neurologic disease.
  • the exemplary target peptides or proteins include, but are not limited to Ab (Amyloid-b peptide), synuclein, fibrin, tau, apolipoprotein E (Apoe), TDP43, prion protein, Huntingtin exon 1, ABri peptide, ADan peptide, fragments of immunoglobulin light chains, fragments of immunoglobulin heavy chains, full or N-terminal fragments of serum amyloid A protein (SAA), transthyretin (TTR), b 2 -h ⁇ ek3 ⁇ 41oI>u1 ⁇ h, N-terminal fragments of apolipoproteinA-I (ApoAI), C-terminal extended apolipoprotein A-II (ApoAII), N-terminal fragments of apolipoprotein A-IV (ApoAIV), apolipo
  • the neurological therapeutic agent may target a peptide or a protein specifically or non-specifically.
  • specific targeting or “specifically targets” refer to an ability to discriminate between possible peptides or proteins in the environment in which the interaction between the neurological therapeutic agent and the target is to occur.
  • a neurological therapeutic agent that interacts, e.g., preferentially interacts, with one particular peptide or protein when other potential neurological therapeutic agents are present is said to "specifically target" the peptide or protein with which it interacts.
  • specific targeting is assessed by detecting or determining the degree of association between the neurological therapeutic agent and its target.
  • specific targeting is assessed by detecting or determining ability of the neurological therapeutic agent to compete with an alternative interaction between its target and another entity. In some embodiments, specific targeting is assessed by performing such detections or determinations across a range of concentrations. Exemplary specific targeting includes, but is not limited to, specific binding of an antibody to its target protein.
  • Exemplary non-specific targeting includes, but is not limited to, the interaction between a small molecule and a class of proteins or peptides.
  • a tyrosine kinase inhibitor may non- specifically inhibit several protein tyrosine kinases.
  • a neurological therapeutic agent of the invention may modulate, e.g., increase or decrease, the activity (e.g, enzyme activity) of its target protein or proteins.
  • a neurological therapeutic agent may modulate, e.g., increase or descrease, the folding and/or aggregation a peptide or protein.
  • a neurological therapeutic agent may binds to a protein, e.g., amyloid-b or tau protein, to reduce the misfolding and aggregation thereof.
  • a neurological therapeutic agent may modulate a target peptide or protein directly or indirectly.
  • the neurological therapeutic agent may interact with the target peptide or protein directly, e.g. , an antibody binds to the target peptide or protein.
  • the neurological therapeutic agent may indirectly modulate the target peptide or protein by interacting with another peptide or protein, and modulate the target by interacting with the another peptide or protein.
  • a protein kinase inhibitor may interact with a protein kinase to indirectly modulate the phosphorylation status of the target peptide or protein.
  • a neurological therapeutic agent may also not target a peptide or protein that is involved in the genesis of a proteinopathy.
  • a neurological therapeutic agent of the invention may prevent or reduce the downstream event of the proteinopathy, such as neuroinflammation that is associated with a neurodegenerative disease.
  • the neurological therapeutic agent comprises a small molecule.
  • small molecule refers to a low molecular weight (e.g., ⁇ 900 daltons) organic compound.
  • Exemplary small molecules that can be used as neurological therapeutic agents include, but are not limited to, Donepezil, Galantamine, Rivastigmine, Memantine, Lanabecestat, Atabecestat, Verubecestat, Elenbecestat, Semagacestat, Tarenflurbil, Brexipiprazole, AXS-05 (Axsome Therapeutics), AC-1204 (Accera), masitinib, amilomotide, guanfacine hydrochloride, octohydroaminoacridine succinate, lumateperone tosylate, AVP-786 (Avanir Pharmaceuticals), ALZT-OP1, AZD-1080 (AstraZeneca), ASN 120290 ( Asceneuron SA), GV
  • a small molecule neurological therapeutic agent may exert the therapeutic effects through various mechanisms.
  • the small molecule neurological agent may reduce mitochondrial dysfunction and ROS production, protein oxidation, lipid peroxidation, nitrosative stress, protein aggregation, amyloidopathy, tauopathy, DNA damage, depletion of endogenous antioxidant enzymes, proteosomeal dysfunction, microglial activation, neuroflammation, and/or neuroepigenetic modification.
  • a small molecule neurological therapeutic agent may include b2 -adrenergic agonists, c-Abl inhibitors, cholinesterase inhibitors, leucine-rich repeat kinase 2 inhibitors, glucocerebrosidase inhibitors, glycogen synthase kinase 3b inhibitors, N-acetylglucosaminidase inhibitors, O-GlcNAcase inhibitors, or anti-inflammatory compounds.
  • the neurological therapeutic agent comprises a protein or peptide.
  • Exemplary proteins or peptides include, but are not limited to, insulin and interferon gamma- lb.
  • the peptide or protein is a chaperone protein or a co-chaperone that facilitates the proper folding of a target peptide or protein. Chaperone proteins assist the conformational folding or unfolding and the assembly or disassembly of other proteins.
  • Exemplary chaperone proteins include, but are not limited to, heat shock proteins (e.g., Hspl04, Hsp90, Hsp70, Hsp27), aB-crystallin, clusterin, a2 -macroglobulin, haptoglobin, human tetrameric transthyretin, proSAAS, protein 7B2, ERdj3/DNAJBl 1, GRP78/BiP, GRP94, GRP170, calnexin, calreticulin, and protein disulfide isomerase.
  • Co-chaperones are proteins that assist chaperones in protein folding and other functions. Exemplary co-chaperones include, but are not limited to J-proteins, DnaJ, Hsp40, DNAJC5, auxilin, RME-8, and Ahal.
  • the neurological therapeutic agent comprises a vaccine.
  • a vaccine is a composition that provides active acquired immunity to a particular disease, such as a neurodegenerative disease, e.g., Alzheimer’s disease.
  • a vaccine typically contains a protein or a peptide that may be disease specific (expressed exclusively by the diseased cell) or disease associated (expressed preferentially by the diseased cell).
  • a vaccine can typically include an adjuvant. The vaccine stimulates the body's immune system to recognize the target and to eliminate or reduce the effect of the target.
  • a vaccine may be directed to amyloid-beta and stimulate the immune system to generate active acquired immunity, e.g., specific antibodies or T cells that recognize amyloid-beta and reduce or eliminate the formation of amyloid plaque.
  • Vaccines can be prophylactic or therapeutic.
  • a vaccine may also be a nucleic acid encoding a protein or a peptide or extract from diseased cells. Exemplary vaccines used in neurodegenerative diseases include, but are not limited to AN-1792.
  • a vaccine may be a nucleic acid vaccine, e.g. , DNA vaccine or RNA vaccine.
  • the neurological therapeutic agent is a nucleic acid or polynucleotide.
  • the nucleic acids or polynucleotides of the invention may include deoxynucleotides, ribonucleotides, modified deoxynucleotides, modified ribonucleotides (e.g., chemical modifications, such as modifications that alter the backbone linkages, sugar molecules, and/or nucleic acid bases), and artificial nucleic acids.
  • the polynucleotide includes, but is not limited to, genomic DNA, cDNA, peptide nucleic acids (PNA) or peptide oligonucleotide conjugates, locked nucleic acids (LNA), bridged nucleic acids (BNA), polyamides, triplex forming oligonucleotides, modified DNA, antisense DNA oligonucleotides, tRNA, mPvNA, rPvNA, modified RNA, miRNA, gRNA, and siRNA or other RNA or DNA molecules.
  • PNA peptide nucleic acids
  • LNA locked nucleic acids
  • BNA bridged nucleic acids
  • polyamides polyamides
  • the polynucleotide of the invention comprises a sequence that encodes a peptide or a protein, e.g., chaperone protein, antibody, or a protein or peptide used in a vaccine, disclosed herein.
  • the nucleic acid or polynucleotide is an inhibitory polynucleotide that inhibits the expression of a gene, e.g., the APP gene that encodes amyloid b protein, or the MAPT gene that encodes the Tau protein.
  • the inhibitory polynucleotide may be RNAi, shRNA, siRNA, or antisense RNA.
  • the polynucleotides of the invention may be expressed from transcription units inserted into DNA or RNA vectors (see, e.g. , Couture, A, et al. , TIG. (1996), 12:5-10; WO 00/22113, WO 00/22114, and US 6,054,299). In some embodiments, expression is sustained (months or longer), depending upon the specific construct used and the target tissue or cell type.
  • These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, el al. , (1995) Proc. Natl. Acad. Sci. USA 92:1292).
  • the individual strand or strands of a polynucleotide encoding a neurological therapeutic agent comprising a nucleic acid molecule can be transcribed from a promoter in an expression vector.
  • Expression vectors are generally DNA plasmids or viral vectors.
  • Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can be used to produce recombinant constructs for the expression of a neurological therapeutic agent as described herein.
  • Delivery of the neurological therapeutic agent expressing vector can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell., e.g., intracerebroventricularly (i.c.v.) or intra-cistema magna (ICM) .
  • intracerebroventricularly i.c.v.
  • ICM intra-cistema magna
  • Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc. ; (c) adeno- associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picomavirus vectors; (i) pox virus vectors such as an orthopox, e.g. , vaccinia virus vectors or avipox, e.g.
  • the constructs can include viral sequences for transfection, if desired.
  • the construct can be incorporated into vectors capable of episomal replication, e.g. EPV and EBV vectors.
  • Constructs for the recombinant expression of a protein of the invention will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the protein in target cells. Other aspects to consider for vectors and constructs are known in the art.
  • Vectors including those derived from retroviruses such as lentivirus, are suitable tools to achieve long- term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells.
  • examples of vectors include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
  • the expression vector may be provided to a cell in the form of a viral vector.
  • Viral vector technology is well known in the art, and described in a variety of virology and molecular biology manuals.
  • Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, and lenti viruses.
  • a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers.
  • the virus is adeno-associated viruses.
  • AAV- mediated delivery of transgene is described elsewhere herein.
  • Expression of natural or synthetic nucleic acids is typically achieved by operably linking a nucleic acid encoding the gene of interest to a promoter, and incorporating the construct into an expression vector.
  • the vectors can be suitable for replication and integration in eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for expression of the desired nucleic acid sequence.
  • Additional promoter elements e.g., enhancing sequences, regulate the frequency of transcriptional initiation.
  • these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another.
  • tk thymidine kinase
  • the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.
  • individual elements can function either cooperatively or independently to activate transcription.
  • a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence.
  • CMV immediate early cytomegalovirus
  • This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto.
  • Another example of a suitable promoter is Elongation Growth Factor- la (EF-la).
  • constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein- Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter.
  • SV40 simian virus 40
  • MMTV mouse mammary tumor virus
  • HSV human immunodeficiency virus
  • LTR long terminal repeat
  • MoMuLV promoter MoMuLV promoter
  • an avian leukemia virus promoter an Epstein- Barr virus immediate early promoter
  • Rous sarcoma virus promoter as well as human gene promoters such as, but
  • the present invention should not be limited to the use of constitutive promoters.
  • Inducible promoters are also contemplated as part of the invention.
  • the use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired.
  • Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.
  • the expression vector to be introduced can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors.
  • the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate transcriptional control sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic -resistance genes, such as neo and the like.
  • Reporter genes may be used for identifying potentially transfected cells and for evaluating the functionality of transcriptional control sequences.
  • a reporter gene is a gene that is not present in or expressed by the recipient source and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.
  • Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei el al, 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially.
  • the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.
  • the methods to deliver a polynucleotide or a nucleic acid to a cell are known in that art.
  • the delivery of the nucleic acid neurological therapeutic agent of the invention to a cell may be achieved in a number of different ways.
  • delivery may be performed by contacting a cell with a nucleic acid of the invention either in vitro, ex vivo, or in vivo.
  • In vivo delivery may be performed directly by administering a composition comprising a peptide or protein neurological therapeutic agent to a subject.
  • in vivo delivery may be performed indirectly by administering one or more vectors that encode and direct the expression of the neurological therapeutic agent.
  • any method of delivery of a nucleic acid of the invention may be adapted for use with the nucleic acid of the invention (see e.g., Akhtar S. and Julian RL., (1992) Trends Cell. Biol. 2(5): 139-144 and WO94/02595, which are incorporated herein by reference in their entireties).
  • factors to be considered for delivering a nucleic acid of the invention include, for example, biological stability of the neurological therapeutic agent, prevention of non-specific effects, and accumulation of the neurological therapeutic agent in the target tissue.
  • the non-specific effects of a neurological therapeutic agent can be minimized by local administration, for example, by direct injection or implantation into a tissue or topically administering a composition comprising the neurological therapeutic agent.
  • Local administration to a treatment site maximizes local concentration of the neurological therapeutic agent, limits the exposure of the neurological therapeutic agent to systemic tissues that can otherwise be harmed by the neurological therapeutic agent or that can degrade the neurological therapeutic agent, and permits a lower total dose of the neurological therapeutic agent to be administered.
  • nucleic acid of the invention for administering a nucleic acid of the invention systemically for the treatment of a disease, such as a neurodegenerative disease, can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of the nucleic acid molecule by endo- and exo-nucleases in vivo. Modification of a nucleic acid molecule also permits targeting of the nucleic acid to a target tissue and avoidance of undesirable off-target effects.
  • a nucleic acid molecule of the invention may be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation.
  • a nucleic acid of the invention may be delivered using a drug delivery system such as a nanoparticle, a dendrimer, a polymer, a liposome, or a cationic delivery system.
  • a drug delivery system such as a nanoparticle, a dendrimer, a polymer, a liposome, or a cationic delivery system.
  • Positively charged cationic delivery systems facilitate binding of nucleic acid molecule (e.g., negatively charged molecule) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of a nucleic acid molecule by the cell.
  • Cationic lipids, dendrimers, or polymers can either be bound to a nucleic acid, or induced to form a vesicle or micelle (see e.g., Kim SH.
  • Some non-limiting examples of drug delivery systems useful for systemic delivery of a nucleic acid of the invention include DOTAP (Sorensen, DR., et al (2003), supra;
  • a nucleic acid e.g., DNA, or mRNA
  • cyclodextrin for systemic administration.
  • Methods for administration and pharmaceutical compositions comprising cyclodextrins may be found in U.S. Patent No. 7,427,605, the entire contents of which are incorporated herein by reference.
  • the nucleic acid of the invention may be incorporated into pharmaceutical compositions suitable for administration.
  • Such compositions typically include one or more species of nucleic acid and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral, or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration.
  • the route and site of administration may be chosen to enhance delivery or targeting of the nucleic acid of the invention to a particular location.
  • intravenous, i.c.v, or ICM injection may be used.
  • Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • Coated condoms, gloves and the like may also be useful.
  • compositions for oral administration include powders or granules, suspensions or solutions in water, syrups, elixirs or non-aqueous media, tablets, capsules, lozenges, or troches.
  • carriers that can be used include lactose, sodium citrate and salts of phosphoric acid.
  • Various disintegrants such as starch, and lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets.
  • useful diluents are lactose and high molecular weight polyethylene glycols.
  • the nucleic acid compositions can be combined with emulsifying and suspending agents. If desired, certain sweetening or flavoring agents can be added.
  • compositions for administration may include sterile aqueous solutions which may also contain buffers, diluents, and other suitable additives.
  • Formulations for may include sterile aqueous solutions which may also contain buffers, diluents, and other suitable additives.
  • the total concentration of solutes may be controlled to render the preparation isotonic.
  • the administration of a nucleic acid of the invention is parenteral, e.g., intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral, or ocular.
  • Administration can be provided by the subject or by another person, e.g., a health care provider.
  • the composition may be provided in measured doses or in a dispenser which delivers a metered dose. Selected modes of delivery are discussed in more detail below.
  • the neurological therapeutic agent comprises a cell.
  • a variety of cells e.g., immune cells (such as T cell specifically targeting amyloid plaque, B cell producing antibody specifically binding amyloid, T reg cell reducing inflammatory reaction), may be used as a neurological therapeutic agent, including, fresh samples derived from subjects, primary cultured cells, immortalized cells, cell-lines, hybridomas, etc.
  • the cells to be used as a neurological therapeutic agent may also include stem cells, such as embryonic stem cells, induced pluripotent stem cells, mobilized peripheral blood stem cells. The cells may be used for various therapeutic applications.
  • the cell may be genetically engineered to express a neurological therapeutic agent, e.g., a protein, a peptide, an antibody, or an inhibitory RNA of the invention.
  • a neurological therapeutic agent e.g., a protein, a peptide, an antibody, or an inhibitory RNA of the invention.
  • the neurological therapeutic agent comprises an antibody.
  • the antibody of the invention binds specifically to a peptide or a protein that forms pathological protein aggregate.
  • a peptide or protein includes, but is not limited to amyloid precursor protein, amyloid beta, fibrin, tau, apolipoprotein E (Apoe), alpha-synuclein, TDP43, and huntingtin.
  • the neurological therapeutic agent can comprise, consist essentially of, or consist of an antibody selected from the group consisting of: bapineuzumab, gantenerumab, aducanumab, solanezumab, crenezumab, pepinemab, ozanezumab, lecanemab, ABT-099, AT-1501, BIIB054, and PRX002.
  • the neurological therapeutic agent can comprise, consist essentially of, or consist of an antibody that binds specifically to a protein associated with AD, for example an antibody (such as a human or humanized antibody) that binds specifically to amyloid beta, for example, bapineuzumab, gantenerumab, aducanumab, solanezumab, immunoglobulin, BAN2401, semorinemab, zagotenemab, and/or crenezumab.
  • example small molecules that inhibit aggregation include apomorphine and carbenoxolone.
  • the flow modulator when a flow modulator and a neurological therapeutic agent are both administered to the subject, unless stated otherwise, the flow modulator will be understood to be a different molecule than the neurological therapeutic agent.
  • the neurological therapeutic agent comprises, consists essentially of, or consist of an antibody or binding fragment selected from the group consisting of bapineuzumab, gantenerumab, aducanumab, solanezumab, and crenezumab.
  • exemplary antibodies include, but are not limited to, ABBV-8E12 (AbbVie), Gosuranemab (BIIB092, IPN007, Bristol- Myers Squibb), PHF1, MCI, DA31, 4E6G7, 6B2G12, TOMA, PHF6, PHF13, HJ9.3, HJ9.4, HJ8.5, 43D, 77E9, AT8, MAb86, pS404 mAb IgG2, pS409-tau, Armanezumab, PHF1, Ta9, Ta4, Tal505, and DC8E8 (see. Jadhav et al.
  • a vaccine may be used as the neurological therapeutic agent.
  • exemplary vaccines include, but are not limited to, Tau 379-408, Tau 417-426, Tau 393-408, Tau 379-408, Tau 195-213, Tau 207- 220, Tau 224-238, Tau aa 395-406, Human paired helical fdaments (PHF’s) Tau 229-237, Tau 199- 208, Tau 209-217, Tau 294-305, and Tau 379-408 (see, Jadhav et al, supra).
  • exemplary antibodies include, but are not limited to, 5B8 as described in Ryu et al., Fibrin-targeting immunotherapy protects against neuroinflmmation and neurodegeneration, Nature Immunology 19, 1212-1223 (2016).
  • exemplary antibodies include, but are not limited to HAE4 as described in Liao et al., Targeting of nonlipidated, aggregated apoE with antibodies inhibits amyloid accumulation, J. of Clin. Invest., 128(5): 2144- 2155.
  • the antibody may be an antibody that binds specifically to semaphorin-4D, for example pepinemab, or an antibody that binds specifically to huntingtin, for example, the antibodies disclosed in US20170166631.
  • the antibody binds specifically to neurite outgrowth inhibitor A (e.g., ozanezumab) or to CD40L (e.g., AT-1501 (Anelixis)).
  • the neurological therapeutic agent may be an antibody binds specifically to alpha-synuclein.
  • anti-alpha-synuclein antibodies include, but are not limited to, BIIB054 (Biogen), PRX002/RG7935 (Roche), prasinezumab (Roche), PD-1601 (AbbVie), 1H7, 5C1, A1-A6, 9E4, 274, NbSyn87*PEST, NAC32, NAC1, AC14, VH14*PEST, syn303, AB1, Human single-chain Fv D10, D5, syn-Ol, syn- 02, syn-04, mAb47, syn-IOH, syn-Fl, syn-F2, LS4-2G12 (see.
  • the antibody may be cinpanemab, ABBV-0805 (AbbVie).
  • the neurological therapeutic agent comprises, consists essentially of, or consists of an antibody or binding fragment selected from the group consisting of bapineuzumab, gantenerumab, aducanumab, solanezumab, and crenezumab, pepinemab, ozanezumab, AT-1501, BIIB054, and PRX002.
  • exemplary antibodies include, but are not limited to, the antibodies disclosed in Patent Nos. US 10202443, US8940872, or Patent Publication Nos. WO2018218352, WO2019134981, (incorporated herein by reference), antibody 3B12A (disclosed in Scientific Reports, 8:6030 (2016), DOI:10.1038/s41598- 018-24463-3).
  • the antibody of the present invention includes bispecific antibodies of multispecific antibodies.
  • Bispecific antibodies or multispecific antibodies include recognize more two or more epitopes. The two or more epitopes may be located on a same protein or on different proteins.
  • Exemplary bispecific or multispecific antibodies include, but are not limited to bispecific monoclonal antibodies to inhibit BACE1 and MAPT for Alzheimer's Disease developed by Denali Therapeutic Inc.
  • the base structure of an antibody is a tetramer, which includes two heavy chains and two light chains. Each chain comprises a constant region, and a variable region. Generally, the variable region is responsible for binding specificity of the antibody. In a typical antibody, each variable region comprises three complementarity determining regions (CDRs) flanked by four framework regions. As such, a typical antibody variable region has six CDRs (three heavy chain CDRs, three light chain CDRs), some or all of which are generally involved in binding interactions by the antibody. The CDRs can be numbered according to an art-recognized method, for example the methodology of Rabat (Rabat, etal.
  • an antibody that “specifically” binds (or binds “specifically”) to an antigen has its ordinary and customary meaning as would be understood by one of ordinary skill in the art in view of this disclosure. It refers to the antibody preferentially binding to the antigen compared to one or more other substances.
  • an antibody that specifically binds to amyloid beta of some embodiments binds to amyloid beta with a numerically lower dissociation constant (R D ) than to other substances present in the CNS.
  • an antibody that specifically binds to amyloid beta binds with a R D that is less than or equal to 1000 nM, 500 nM, 200 nM, 100 nM, 75 nM, 50 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 2 nM, 1 nM, 500 pM, 100 pM, 75 pM, 50 pM, 25 pM, 10pm, or 5 pM, including ranges between any two of the listed values.
  • a R D for a particular antigen can be determined, for example, by surface plasmon resonance, for example using a BIACORE apparatus.
  • a neurological therapeutic agent as in accordance with methods, compositions, and uses of embodiments herein can comprise, consist essentially of, or consist of any of a number of antibodies.
  • Example monoclonal amyloid beta antibodies that can be used as a neurological therapeutic agents in accordance with embodiments herein include bapineuzumab, gantenerumab, aducanumab, solanezumab, and crenezumab. Solanezumab and crenezumab bind to a helix-beta coil epitope in the midsection of amyloid beta, while bapineuzumab, gantenerumab, and aducanumab bind to the N-terminal region of amyloid beta.
  • a neurological therapeutic agent comprises, consists essentially of, or consists of an antibody selected from the group consisting of bapineuzumab, gantenerumab, aducanumab, solanezumab, and crenezumab, or an antigen binding fragment of any of the listed antibodies.
  • a neurological therapeutic agent comprises, consists essentially of apomorphine or carbenoxolone.
  • a neurological therapeutic agent comprises, consists essentially of, or consists of an antibody selected from the group consisting of bapineuzumab, gantenerumab, aducanumab, solanezumab, and crenezumab, or an antigen binding fragment of any of the listed antibodies, or apomorphine or carbenoxolone.
  • a neurological therapeutic agent comprises, consists essentially of, or consists of an antibody or antigen binding fragment that comprises a heavy chain variable region and a light chain variable region of any one of bapineuzumab, gantenerumab, aducanumab, solanezumab, or crenezumab.
  • a neurological therapeutic agent comprises, consists essentially of, or consists of an antibody or antigen binding fragment that comprises a heavy chain variable region and a light chain variable region that are, respectively, at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a heavy chain variable region and light chain variable of bapineuzumab, gantenerumab, aducanumab, solanezumab, or crenezumab.
  • a neurological therapeutic agent comprises, consists essentially of, or consists of an antibody or antigen binding fragment that comprises a light chain variable region that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a light chain variable region of bapineuzumab, gantenerumab, aducanumab, solanezumab, or crenezumab.
  • the antibody or antigen binding fragment can further comprise a heavy chain variable region of the noted antibody (bapineuzumab, gantenerumab, aducanumab, solanezumab, or crenezumab).
  • a neurological therapeutic agent comprises, consists essentially of, or consists of an antibody or antigen binding fragment that comprises a HCDR1, a HCDR2, and a HCDR3 of a heavy chain variable region, and a LCDR1, a LCDR2, and a LCDR3 of a light chain variable region of any one of bapineuzumab, gantenerumab, aducanumab, solanezumab, or crenezumab (that is, the antibody or antigen binding fragment comprises the noted six CDR’s of any one of the listed antibodies).
  • the neurological therapeutic agent comprises, consists essentially of, or consist of an antibody selected from the group consisting of bapineuzumab, gantenerumab, aducanumab, solanezumab, and crenezumab.
  • the neurological therapeutic agent comprises a chimeric antigen receptor T-cell (CAR-T cell) that binds specifically to a protein associated with the proteinopathy of the patient.
  • the neurological disease or disorder comprises a tauopathy and/or an amyloidosis such as AD
  • the neurological therapeutic agent comprises a chimeric antigen receptor T-cell (CAR-T cell) that binds specifically to an amyloid beta protein.
  • the CAR-T cell can comprise a chimeric antigen receptor comprising a heavy chain variable region and a light chain variable region of any of the antibodies described herein.
  • the CAR-T cell can comprise a chimeric antigen receptor comprising a HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of any of the antibodies described herein.
  • a number of approaches are available for producing suitable antibodies that specifically bind to a target peptide or protein, e.g., amyloid beta, a-synuclein, fibrin, tau, apolipoprotein E (Apoe), or TDP43, in accordance with methods and uses of embodiments herein.
  • a target peptide or protein e.g., amyloid beta, a-synuclein, fibrin, tau, apolipoprotein E (Apoe), or TDP43
  • a host organism is immunized with an antigen comprising, consisting essentially of, or consisting of an amyloid beta, for example amyloid precursor protein (APP) or a fragment thereof.
  • an antigen comprising, consisting essentially of, or consisting of an amyloid beta, for example amyloid precursor protein (APP) or a fragment thereof.
  • APP amyloid precursor protein
  • a sequence of APP is available as Uniprot accession no. P56199 (SEQ ID NO: 5
  • a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 5 sequence can be used to immunize a host in order to produce antibodies that bind specifically to amyloid beta in accordance with some embodiments.
  • the host organism can be a non-human mammal such as a mouse, rat, guinea pig, rabbit, donkey, goat, or sheep.
  • Isolated antibody-producing cells can be obtained from the host organism, and the cells (or antibody-encoding nucleic acids thereof) can be screened for antibodies that binds specifically to amyloid beta.
  • antibody- producing cells are immortalized using hybridoma technology, and the resultant hybridomas are screened for antibodies that bind specifically to amyloid beta.
  • antibody encoding nucleic acids are isolated from antibody-producing cells, and screened for antibodies that bind specifically to amyloid beta.
  • An example protocol for screening human B cell nucleic acids is described in Huse et al. , Science 246: 1275-1281 (1989), which is hereby incorporated by reference in its entirety.
  • nucleic acids of interest are identified using phage display technology (See, e.g., Dower et al, WO 91/17271 and McCafferty et al, WO 92/01047, each of which is hereby incorporated by reference in its entirety).
  • Phage display technology can also be used to mutagenize variable regions (or portions thereof such as CDRs) of antibodies previously shown to have affinity for amyloid beta. Variant antibodies can then be screened by phage display for antibodies having desired affinity to amyloid beta.
  • the antibody that specifically binds to amyloid beta is formatted as an antigen binding fragment (which may be referred to herein simply as a “binding fragment”).
  • Example antigen binding fragments suitable for methods and uses of some embodiments can comprise, consist essentially of, or consist of a construct selected from the group consisting of Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments; Fd fragment; minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3- FR4 peptide; minibodies; diabodies; and single-chain fragments such as single-chain Fv (scFv) molecules.
  • CDR complementarity determining region
  • bispecific or multispecific antibodies or antigen binding fragments are also contemplated in accordance with methods and uses of some embodiments.
  • Other engineered molecules such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "antigen-binding fragment," as used herein.
  • an “antibody” is mentioned herein, a binding fragment of that antibody is also contemplated.
  • the amyloid beta antibody is chimeric, human, or humanized. In some embodiments, the amyloid beta antibody is human, or humanized.
  • the host comprises genetic modifications to produce or facilitate the production of human immunoglobulins.
  • XenoMouseTM mice were engineered with fragments of the human heavy chain locus and kappa light chain locus, respectively, which contained core variable and constant region sequences (described in detail Green et al. Nature Genetics 7: 13-21 (1994), which is hereby incorporated by reference in its entirety).
  • mice have been engineered to produce antibodies comprising a human variable regions and mouse constant regions. The human heavy chain and light chain variable regions can then be reformatted onto a human constant region to provide a fully human antibody (described in detail in U.S. Pat. No.
  • a host monoclonal antibody is formatted as a chimeric antibody or is humanized, so that the antibody comprises at least some human sequences.
  • an approach for producing humanized antibodies can comprise CDR grafting.
  • an antigen can be delivered to a non-human host (for example a mouse), so that the host produces antibody against the antigen.
  • monoclonal antibody is generated using hybridoma technology.
  • V gene utilization in a single antibody producing cell of the host is determined. The CDR’s of the host antibody can be grafted onto a human framework.
  • V genes utilized in the non-human antibody can be compared to a database of human V genes, and the human V genes with the highest homology can be selected, and incorporated into a human variable region framework. See, e.g., Queen, U.S. Pat. No. 5,585,089, which is hereby incorporated by reference in its entirety.
  • Isolated oligonucleotides encoding an antibody of interest can be expressed in an expression system, such as a cellular expression system or a cell-free system in order to produce an antibody that binds specifically to amyloid beta in accordance with methods and uses of embodiments herein.
  • exemplary cellular expression systems include yeast (e.g., mammalian cells such as CHO cells or BHK cells, E.
  • yeast expression vectors containing the nucleotide sequences encoding antibodies
  • insect cell systems infected with recombinant virus expression vectors e.g., baculovirus
  • plant cell systems infected with recombinant virus expression vectors e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV
  • recombinant plasmid expression vectors e.g., Ti plasmid
  • mammalian cell systems e.g., COS, CHO, BHK, 293, 3T3 harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses.
  • a neurological therapeutic agent of the invention may be used in combination with one or more different neurological therapeutic agents of the invention.
  • a small molecule neurological therapeutic agent may be used in combination with an antibody therapeutic agent.
  • Different neurological therapeutic agents may be formulated in a same pharmaceutical composition or in different pharmaceutical compositions. Different neurological therapeutic agents may be administered concurrently or sequentially.
  • the neurological disease is a proteinopathy.
  • the neurological disease comprises a proteinopathy as described herein (e.g., a tauopathy and/or amyloidosis such as AD).
  • a tauopathy and/or amyloidosis such as AD.
  • Such diseases can occur in subjects, for example humans, as well as non-human animals, such as non-human mammals, and non-human primates in some embodiments.
  • a neurological disease such as a neurodegenerative, neurodevelopmental, neuroinflammatory, or neuropsychiatric disease associated with accumulation of macromolecules, cells, and debris in the CNS is treated, prevented, inhibited, or reduced by methods, uses, or compositions that increase flow, drainage, and/or clearance in meningeal lymphatic vessels.
  • neurodegenerative, neurodevelopmental, neuroinflammatory, or neuropsychiatric diseases associated with accumulation of macromolecules, cells, and debris in the CNS are treated, prevented, inhibited, or reduced by methods, uses, or compositions that counteract the effects (e.g., changes in the hippocampal transcriptome) of decreased flow with or without restoring flow.
  • neurological diseases associated with accumulation of macromolecules, cells, and debris in the CNS are treated, prevented, inhibited, or reduced. Examples of neurological diseases include proteinopathies, for example tauopathies and/or amyloidosis such as AD (e.g., familial AD and/or sporadic AD).
  • AD Alzheimer's disease
  • dementia dementia
  • age-related dementia dementia
  • PD cerebral edema
  • ALS dementia
  • hemorrhagic stroke ASD
  • epilepsy Down’s syndrome
  • HCHWA-D Familial Danish/British dementia
  • DLB LB variant of AD
  • MSA FENIB
  • FTD FTD
  • HD Kennedy disease/SBMA
  • DRPLA DRPLA
  • SCAtype I SCA2, SCA3 (Machado-Joseph disease)
  • SCA6, SCA7, SCA17, CJD (such as familial CJD), Kuru, GSS, FFI, CBD, PSP, CAA, AIDS-related dementia complex, or a combination of two or more of the listed items.
  • neurological diseases can include AD, dementia, age-related dementia, PD, cerebral edema, ALS, PANDAS, meningitis, hemorrhagic stroke, ASD, and epilepsy.
  • the neurological disease comprises, consists essentially of, or consists of a proteinopathy, for example AD (such as familial AD and/or sporadic AD), Down’s syndrome, HCHWA-D, Familial Danish/British dementia, PD, DLB, LB variant of AD, MSA,
  • neurological diseases associated with amyloid beta for example an amyloidosis such as AD (e.g., familial AD and/or sporadic AD) in the CNS are treated, prevented, inhibited, or reduced by methods, uses, or compositions that counteract the effects of decreased flow with or without restoring flow.
  • AD amyloidosis
  • the neurological disease for example a proteinopathy (such as a tauopathy and/or an amyloidosis, e.g., AD) can be prevented, treated, or ameliorated prophylactically.
  • a subject having one or more risk factors for the neurological disease can be determined to be in need of receiving a method, use, or composition described herein.
  • a subject may have accumulated amyloid beta plaques in their CNS, and may benefit from increased flow, increased drainage, increased clearance and/or reduction of amyloid beta plaques, even if they do not yet have an neurological disease diagnosis based on cognitive symptoms.
  • a number of risk factors for AD are suitable as risk factors in accordance with methods, compositions, and uses of some embodiments herein, for example familial AD, a genetic marker for AD, or a symptom of AD such as early dementia.
  • the foremost risk factor for sporadic AD is age.
  • increased risk of this form of AD has also been attributed to diverse genetic abnormalities.
  • One of them is diploidy for apolipoprotein-Ee4 (Apo-Es4), widely viewed as a major genetic risk factor promoting both early onset of amyloid beta aggregation and defective amyloid beta clearance from the brain (Deane et al, 2008; Zlokovic, 2013).
  • the risk factor for AD is selected from the group consisting of at least one of the following: diploidy for apolipoprotein-E-epsilon-4 (apo-E-epsilon-4), a variant in apo-J, a variant in phosphatidylinositol-binding clathrin assembly protein (PICALM), a variant in complement receptor 1 (CR3), a variant in CD33 (Siglee-3), or a variant in triggering receptor expressed on myeloid cells 2 (TREM2), age, or a symptom of dementia.
  • apo-E-epsilon-4 diploidy for apolipoprotein-E-epsilon-4
  • PICALM phosphatidylinositol-binding clathrin assembly protein
  • CR3 complement receptor 1
  • Siglee-3 a variant in CD33
  • TREM2 myeloid cells 2
  • the invention is based upon, at least in part, the surprising discovery that a subject has degeneration of lymphatic vasculature in the central nervous system of the subject prior to the onset of the neurological disease.
  • degeneration of lymphatic vasculature refers to the reduction or loss or lymphatic vessel coverage (in area) in the central nervous system. The reduced coverage may be cause by the reduced length, the diameter, and/or branching point of lymphatic vessels.
  • the degeneration of lymphatic vessel occurs at the superior sagittal sinus, dural confluence of sinuses, the transverse (TS), sigmoid (SS), or petrosquamosal (PSS) sinuses.
  • the invention provides a method of identifying a subject that has an enhanced risk of developing neurological disease prior to the onset of the neurological disease.
  • the term “enhanced risk,” as used herein, refers to a higher probability to develop certain neurological diseases, e.g., Alzheimer’s disease, as compared to a reference probability (reference risk).
  • the reference risk is the probability of developing such a neurological disease in general population.
  • the method includes detecting the degeneration of lymphatic vasculature in the central nervous system of the subject. Any methods that can be used to detect the degeneration of the lymphatic vasculature in the central nervous system are encompassed in this invention.
  • the detection method is a non-invasive detection method that visualizes the lymphatic vasculature of the central nervous system.
  • exemplary non-invasive detection method includes magnetic resonance imaging as described in Abstinta et al, Human and nonhuman primate meninges harbor lymphatic vessels that can be visualized noninvasively by MRI, eLife 2017: 6: e29738, DOI: 10.7554, incorporated herein by reference.
  • MRI Magnetic resonance imaging
  • a magnetic dye is administered to the subject.
  • the magnetic dye has molecules that are small enough to leak out of blood vessels in the dura into lymphatic vessels, but too big to pass through the blood-brain barrier and enter other parts of the brain.
  • the lymphatic vessels of the central nervous system can be specifically visualized.
  • the lymphatic vasculature can be visualized using in vivo fluorescence imaging method. Exemplary fluorescence imaging in human was described in Piper et al., Toward whole-body fluorescence imaging in humans, PLoS One, 2013; 8(12): e83749, incorporated herein by reference.
  • the degeneration of lymphatic vasculature may be reflected in the decrease of lymphatic coverage by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 99%, or between about 10% to about 99%.
  • this invention is based upon, at least in part, that the gene expression profde in lymphatic endothelia cells or immune cells in the central nervous system is altered in a subject that has an enhanced risk of developing a neurological disease, e.g., Alzheimer’s disease.
  • the alteration in the gene expression profde is the change of gene expression level of one or more genes in Tables 2-29.
  • the present invention provides a method of identifying a subject that has an enhanced risk of developing a neurological disease, e.g., Alzheimer’s disease, prior to the onset of the neurological disease.
  • the method includes detecting the alteration of gene expression level in one or more genes in Table 2-29 in lymphatic endothelial cells (LECs) or immune cells in central nervous system.
  • LECs lymphatic endothelial cells
  • the immune cells may be from the meninges or brain cortices.
  • the gene expression level is “altered,” e.g., increased or depressed as compared to a reference gene expression level.
  • the reference gene expression level may be the gene expression level of a healthy subject who is known to have the reference risk of developing a neurologic disease, e.g., Alzheimer’s disease, or the average gene expression level in a general population.
  • LECs or immune cells from central nervous system may be obtained from biopsy from deep cervical lymph nodes. Single cell RNA sequence may be then performed on the cells to determine the alteration in the gene expression level.
  • the expression level of certain genes may be reduced by about
  • the expression level of certain genes may be increased by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 99%, or between about 10% to about 99%.
  • the expression level of certain genes may be increased by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 99%, between about 10% to about 99%, about one fold, about two fold, about 4 fold, about 8 fold, about 16 fold, about 32 fold, about 50 fold, about 100 fold, or about one to about 100 fold, or more than about 100 fold.
  • this invention is based upon, at least in part, that a subject with enhanced risk of developing a neurological disease, e.g., Alzheimer’s disease, has increased number of immune cell in the central nervous system.
  • a neurological disease e.g., Alzheimer’s disease
  • the present invention provides a method of identifying a subject that has an enhanced risk of developing a neurological disease, e.g. , Alzheimer’s disease, prior to the onset of the neurological disease.
  • the method includes detecting the increase in the number of immune cells in the central nervous system.
  • the number of immune cells increases if more immune cells are identified in the central nervous system as compared to a reference number of immune cells.
  • the reference number of immune cells is a number of immune cells in the central nervous system of a healthy subject who is known to have a reference risk or the number of immune cells in the central nervous system in a general population.
  • the immune cells are CD45 hlgh microglia or recruited lymphocytes. In some embodiments, the immune cells are CD45 mt microglia or recruited lymphocytes that express H-2KD. In some other embodiments, the immune cells are selected from the group consisting of B cells, CD4 + T cells, CD8 + T cells, and type 3 innate lymphoid cells (ILC3s).
  • the number of immune cells in a subject’s central nervous system may be determined by in vivo fluorescence imaging.
  • an antibody specific to a cell surface protein may be conjugated to a fluorescence entity.
  • the antibody-fluorescence entity complex may be administered to a subject.
  • the fluorescence density may reflect the number of the immune cells.
  • the number of immune cells may be increased by about
  • the present invention is based upon, at least in part, the discovery that certain genes containing neurological disease-associated Single Nucleotide Polymorphism (SNP) are highly expressed in lymphatic endothelial cells. Accordingly, the present invention provides a method of identifying a subject that has an enhanced risk of developing a neurological disease. The method includes detecting one or more single nucleotide polymorphisms as listed in Table 2-29.
  • SNP neurological disease-associated Single Nucleotide Polymorphism
  • the subject is a human subject.
  • the human subject may be about 20 years old, about 30 years old, about 40 years old, about 50 years old, about 60 years old, about 70 years old, about 80 years old, about 90 years old, about 100 years old, or any age between about 20 and about 100 years old.
  • the human subject is previously known to have an enhanced risk of developing a neurologic disease, e.g., Alzheimer’s disease. Such an enhanced risk may be evaluated by investigating the family history of the subject or by genetic screening.
  • the present invention is based upon, at least in part, the surprising discovery that administration of a neurological therapeutic agent, e.g. , an antibody against Ab, prior to the onset of a neurological disease, e.g., Alzheimer’s disease, can reduce the risk of developing such neurological disease.
  • a neurological therapeutic agent e.g., an antibody against Ab
  • the present invention provides a method of reducing the risk, or delaying the onset of a neurological disease.
  • the method includes administering to a subject an effective amount of a neurological therapeutic agent according to the present invention.
  • the subject is identified to have an enhanced risk of developing the neurological disease according to any method disclosed herein.
  • the method of reducing the risk, or delaying the onset of a neurological disease further include administering to the subject an effective amount of flow modulator according to the present invention.
  • compositions for use or uses for increasing flow in fluid in the central nervous system of a subject, or compositions for use in these methods.
  • the components of any of the noted compositions can be provided separately as “product combinations” in which the components are provided in two or more precursor compositions, which can either be combined to form the final composition (e.g., mix a flow modulator with a neurological disease therapeutic agent to arrive at a final composition comprising the flow modulator neurological disease therapeutic agent), or used in conjunction to achieve an effect similar to the single composition (e.g., administer a flow modulator and neurological disease therapeutic agent to a subject simultaneously or sequentially).
  • Some embodiments include a composition or product combination comprising a flow modulator (e.g., VEGFR3 agonist and/or FGF), and a neurological disease therapeutic agent.
  • the neurological disease therapeutic agent can be different from the flow modulator.
  • the composition can be for medical use, for example, for use in treating, preventing, or ameliorating the symptoms of a neurological disease, for example a proteinopathy as described herein (e.g. , a tauopathy and/or amyloidosis such as AD).
  • the methods or uses can include determining whether the subject is in need of increased fluid flow in the central nervous system.
  • the method or use can include administering an effective amount of flow modulator (such as a VEGFR3 agonist and/or FGF2) to a meningeal space of the subject and administering a neurological therapeutic agent to the subject (for example, to the CNS, such as the meningeal space).
  • flow modulator such as a VEGFR3 agonist and/or FGF2
  • a neurological therapeutic agent for example, to the CNS, such as the meningeal space.
  • the flow modulator e.g., VEGFR3 agonist and/or FGF2
  • the flow modulator and neurological therapeutic agent can be administered in the same composition, or in separate compositions as described herein.
  • the amount of flow modulator (e.g., VEGFR3 agonist and/or FGF2) can increase flow for example, by increasing the diameter of a meningeal lymphatic vessel of the subject, by increasing the quantity of meningeal lymphatic vessels of the subject, and/or by increasing drainage through meningeal lymphatic vessels of the subject.
  • VEGFR3 agonist and/or FGF2 can increase flow for example, by increasing the diameter of a meningeal lymphatic vessel of the subject, by increasing the quantity of meningeal lymphatic vessels of the subject, and/or by increasing drainage through meningeal lymphatic vessels of the subject.
  • the neurological therapeutic agent can treat, inhibit, ameliorate, reduce the symptoms of, reduce the likelihood of, or prevent the neurological disease.
  • the neurological therapeutic agent e.g., amyloid beta antibody
  • the flow modulator e.g., VEGFR3 agonist and/or FGF2.
  • the synergy can comprise greater clearance of protein deposits (e.g., amyloid deposits) than either the neurological therapeutic agent or flow modulator on its own, for example at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 57%, 60%, 70%, 80%, or 90% less amyloid plaque density, and/or at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 57%, 60%, 70%, 80%, 90% lower amyloid plaque size compared to either the neurological therapeutic agent or flow modulator alone.
  • the synergy comprises enhancement of memory, and/or delay in the onset or progression of dementia.
  • the fluid comprises cerebral spinal fluid (CSF), interstitial fluid (ISF), or both.
  • the VEGFR3 agonist comprises, consists essentially of, or consists of VEGF-c or VEGF-d or an analog, variant, or fragment thereof. It is also contemplated that for compositions and methods and uses in some embodiments herein, FGF2 can be substituted for the indicated VEGFR3 agonist in order to increase flow, or can be used in addition to a VEGFR3 agonist in order to increase flow.
  • Such methods of, compositions for, or use for increasing fluid flow in the CNS can be useful for treating, preventing, or ameliorating the symptoms of neurological diseases associated with the increased concentration and/or accumulation of molecules, cells or debris in the CNS (e.g., protein deposits such as amyloid deposits), for example in a neurological disease, for example a proteinopathy such as a tauopathy and/or amyloidosis (e.g., AD).
  • a subject can be determined to be in need of increased fluid flow by determining whether the subject has a neurological disease, or is at risk of developing a neurological disease.
  • the disease can be associated with the increased concentrations and/or accumulation of molecules or cells or debris in the CNS, for example a proteinopathy such as a tauopathy and/or amyloidosis (e.g., AD).
  • a proteinopathy such as a tauopathy and/or amyloidosis (e.g., AD).
  • the subject can be determined to be at risk for the disease, for example through having familial occurrence of the disease, by having one or more genetic or protein or metabolite markers associated with the disease, through advanced age, or by exhibiting symptoms of the disease, for example early dementia in the case of AD.
  • determining whether the subject is in need of increased fluid flow comprises determining the subject to have a neurological disease, for example a proteinopathy such as a tauopathy and/or amyloidosis (e.g., AD).
  • a neurological disease for example a proteinopathy such as a tauopathy and/or amyloidosis (e.g., AD).
  • determining whether the subject is in need of increased fluid flow comprises determining the subject to have a risk factor for the neurological disease associated with the increased concentration and/or accumulation of molecules or macromolecules or cells or debris in the CNS as described herein. In some embodiments, determining whether the subject is in need of increased fluid flow comprises determining the subject to have a risk factor, and also determining the subject to have the disease itself. In some embodiments, the neurological disease is Alzheimer’s disease, and the risk factor is a risk factor for Alzheimer’s disease as described herein.
  • the flow modulator (e.g., VEGFR3 agonist and/or FGF2) is administered to the subject after determining that the subject has a risk factor for the neurological disease (even if the subject does not necessarily have the disease itself), for example for prophylactic treatment or prevention. In some embodiments, the flow modulator (e.g., VEGFR3 agonist and/or FGF2) is administered to the subject after determining that the subject has the neurological disease.
  • systemic administration is not required for the flow modulator (e.g., VEGFR3 agonist and/or FGF2) to effectively modulate meningeal lymphatic vessel size and drainage, or flow, and/or for the combination of the neurological therapeutic agent and flow modulator to inhibit, treat, reduce the likelihood of, delay the onset of, prevent, or ameliorate symptoms of the neurological disease.
  • the flow modulator e.g., VEGFR3 agonist and/or FGF2
  • the neurological therapeutic agent is administered selectively to the meningeal space of the subject.
  • the flow modulator e.g., VEGFR3 agonist and/or FGF2
  • the neurological therapeutic agent is administered to the subject.
  • the neurological therapeutic agent may be administered to the meningeal space, or to a different location, for example, subcutaneously, intravenously, parenterally, orally, by inhalation, transdermally, or by rectal administration.
  • the flow modulator e.g., VEGFR3 agonist and/or FGF2
  • the neurological therapeutic agent is administered to the meningeal space, but is not administered outside the CNS.
  • the flow modulator e.g. , VEGFR3 agonist and/or FGF2
  • the neurological therapeutic agent is administered to the meningeal space, but is not administered to the blood.
  • the flow modulator e.g., VEGFR3 agonist and/or FGF2
  • the neurological therapeutic agent is administered to the subject by a route selected from the group consisting of at least one of the following: nasal administration, transcranial administration, contact with cerebral spinal fluid (CSF) of the subject, pumping into CSF of the subject, implantation into the skull or brain, contacting a thinned skull or skull portion of the subject with the VEGFR3 agonist and/or FGF2 and/or the neurological therapeutic agent, or expression in the subject of a nucleic acid encoding the VEGFR3 agonist and/or FGF2 and/or the neurological therapeutic agent, or a combination of any of the listed routes.
  • CSF cerebral spinal fluid
  • the VEGFR3 agonist is administered.
  • the VEGFR3 agonist is selected from the group consisting of at least one of the following: VEGF-c, VEGF-d, or an analog, variant, or functional fragment thereof.
  • the neurological therapeutic agent comprises, consists essentially of, or consists of an amyloid beta antibody.
  • the neurological therapeutic agent comprises, consists essentially of, or consists of an amyloid beta antibody.
  • the administration of the flow modulator (VEGFR3 agonist such as VEGF-c, and/or FGF2) and the neurological therapeutic agent (e.g., amyloid beta antibody) results in an increase in CNS fluid flow, meningeal lymphatic vessel diameter, meningeal lymphatic vessel number, meningeal lymphatic vessel drainage, or amelioration of symptoms of a neurological disease.
  • VEGFR3 agonist such as VEGF-c, and/or FGF2
  • the neurological therapeutic agent e.g., amyloid beta antibody
  • the administration of the flow modulator (e.g., VEGFR3 agonist and/or FGF2) and the neurological therapeutic agent increases diameter of the meningeal lymphatic vessel is increased by at least about 5%, for example at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%, including ranges between any two of the listed values.
  • an average diameter of a population of meningeal lymphatic vessels of the subject is increased by a value noted herein.
  • the administration of the flow modulator (e.g., VEGFR3 agonist and/or FGF2) and the neurological therapeutic agent increases fluid flow in the central nervous system of the subject, comprising increasing a rate of perfusion of fluid throughout an area of the subject’s brain.
  • the administration of the flow modulator (VEGFR3 agonist such as VEGF-c, and/or FGF2), and neurological therapeutic agent (e.g., amyloid beta antibody) increases the ISF flow and reduces the quantity and/or average size of amyloid beta plaques in the subject’s CNS.
  • the quantity of accumulated amyloid beta plaques can be reduced by at least 2%, for example, at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, including ranges between any two of the listed values.
  • the average size of accumulated amyloid beta plaques can be reduced by at least 2%, for example, at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, including ranges between any two of the listed values. It is shown herein that some brains of humans with AD have structures resembling amyloid beta plaques in the meninges. Accordingly, in some embodiments, at least some of the accumulated amyloid beta plaques are in the meninges of the subject’s brain.
  • administering the combination of the flow modulator (VEGFR3 agonist such as VEGF-c, and/or FGF2), and the neurological therapeutic agent (e.g., amyloid beta antibody) increases clearance of soluble molecules in the brain of the subject. Clearance of soluble molecules can be ascertained, for example, by monitoring the retention of a particular compound, molecule, or label over an area of the brain over a particular period of time.
  • VEGFR3 agonist such as VEGF-c, and/or FGF2
  • the neurological therapeutic agent e.g., amyloid beta antibody
  • administering the combination of the FGF2 or VEFR3 agonist (e.g., VEGF-c) and the neurological therapeutic agent (e.g., amyloid beta antibody) increases clearance of soluble molecules in the brain of the subject by at least 2%, for example, at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, including ranges between any two of the listed values.
  • Some embodiments include methods, compositions for use, and uses for reducing a quantity of protein aggregates, such as amyloid beta, fibrin, tau, or alpha-synuclein aggregates.
  • the protein aggregate comprises amyloid beta plaques.
  • the present invention provides compositions and methods for reducing accumulated amyloid beta plaques, or decreasing the rate of accumulation of amyloid beta plaques, in a subject having a neurological disease or a risk factor for such a disease, or compositions for use in such methods.
  • the methods or uses can include determining the subject to have the neurological disease or the risk factor.
  • the methods or uses can include administering a flow modulator (e.g. , VEGFR3 agonist and/or FGF2) to a meningeal space of the subject, so that fluid flow (e.g., flow of ISF, CSF, or both) in the central nervous system of the subject is increased, and further administering and a neurological therapeutic agent to the subject (the neurological therapeutic agent can be administered to a meningeal space or to a different location).
  • a flow modulator e.g. , VEGFR3 agonist and/or FGF2
  • the VEGFR3 agonist can comprise (or consist essentially of, or consist of) VEGF-c
  • the neurological therapeutic agent can comprise (or consist essentially of, or consist of) an amyloid beta antibody.
  • at least some of the accumulated amyloid beta plaques are in the meninges of the subject’s brain.
  • the quantity of accumulated amyloid beta plaques, the average size of the accumulated amyloid beta plaques, and/or the rate of accumulation is reduced by at least 2%, for example, at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% including ranges between any two of the listed values.
  • the flow modulator e.g., VEGFR3 agonist and/or FGF2
  • the neurological therapeutic agent is administered selectively to the meningeal space.
  • the flow modulator (e.g., VEGFR3 agonist and/or FGF2) and/or the neurological therapeutic agent is administered to the CNS, but not outside the CNS.
  • the flow modulator (e.g., VEGFR3 agonist and/or FGF2) and/or the neurological therapeutic agent is administered to the CNS, but not blood.
  • the VEGFR3 agonist is selected from the group consisting of at least one of the following: VEGF-c, VEGF-d, or an analog, variant, or functional fragment thereof.
  • the neurological therapeutic agent comprises, consists essentially of, or consists of an amyloid beta antibody.
  • administering the flow modulator (e.g., VEGFR3 agonist and/or FGF2) and/or the neurological therapeutic agent increases the diameter of a meningeal lymphatic vessel of the subject’s brain by at least 2%, for example at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, including ranges between any two of the listed values, thus increasing flow in ISF.
  • increased fluid flow in the central nervous system of the subject comprises an increased rate of perfusion of fluid throughout an area of the subject’s brain.
  • increased fluid flow in the central nervous system of the subject can comprise an increased rate of perfusion out of the subject’s central nervous system.
  • the subject is known to have the neurological disease, for example AD (such as familial AD and/or sporadic AD), Down’s syndrome, HCHWA-D, Familial Danish/British dementia, PD, DEB, FB variant of AD, MSA, FENIB, AES, FTD, HD, Kennedy disease/SBMA, DRPLA; SCA type I, SCA2, SCA3 (Machado-Joseph disease), SCA6, SCA7,
  • AD familial AD and/or sporadic AD
  • Down’s syndrome HCHWA-D
  • Familial Danish/British dementia Familial Danish/British dementia
  • PD DEB
  • FB variant of AD MSA, FENIB, AES, FTD, HD, Kennedy disease/SBMA, DRPLA
  • SCA type I SCA2, SCA3 (Machado-Joseph disease), SCA6, SCA7,
  • the neurological disease can comprise a proteinopathy.
  • the method further includes determining that the subject has the neurological disease. In some embodiments, for example if the method or use is prophylactic, the method comprises determining whether the subject has the risk factor for the neurological disease, even if the subject does not necessarily have a diagnosis for the disease itself.
  • risk factors for AD that are useful in accordance with methods, compositions, and uses of some embodiments herein include diploidy for apolipoprotein-E-epsilon-4 (apo-E-epsilon-4), a variant in apo-J, a variant in phosphatidylinositol-binding clathrin assembly protein (PICALM), a variant in complement receptor 1 (CR3), a variant in CD33 (Siglee-3), or a variant in triggering receptor expressed on myeloid cells 2 (TREM2), familial AD, advanced age, or a symptom of dementia.
  • apo-E-epsilon-4 diploidy for apolipoprotein-E-epsilon-4
  • PICALM phosphatidylinositol-binding clathrin assembly protein
  • CR3 complement receptor 1
  • Siglee-3 a variant in CD33
  • TREM2 myeloid cells 2
  • the present invention provides methods of reducing extracellular protein aggregates, e.g., amyloid plaque, or protein aggregates released by a cell, e.g., a neuron.
  • a neurological therapeutic agent such as an antibody or an anti-aggregation small molecule compound, may binds or interacts with an amyloid plaque to reduce the protein aggregation.
  • the present invention provides methods of reducing intracellular protein aggregates, e.g., a huntingtin aggregate within a cell.
  • a flow modulator of the invention e.g., VEGF-c
  • a neurological therapeutic agent may be delivered into a cell to reduce the formation of the protein aggregate.
  • a small inhibitory RNA may be delivered to a cell to reduce the expression of a protein, e.g., amyloid-beta, thereby reducing the formation of amyloid aggregate in the cell.
  • Some embodiments include a method, use, or composition for use in increasing clearance of molecules (such as proteins, e.g., amyloid beta) from the central nervous system of a subject.
  • the method or use can comprise administering a composition comprising, consisting of, or consisting essentially of a flow modulator (e.g., VEGFR3 agonist and/or FGF2) to a meningeal space of the subject, in which fluid flow in the central nervous system of the subject is increased, and administering a neurological therapeutic agent (e.g., amyloid beta antibody) to the subject.
  • a flow modulator e.g., VEGFR3 agonist and/or FGF2
  • a neurological therapeutic agent e.g., amyloid beta antibody
  • the neurological therapeutic agent may be administered to the meningeal space, or to a different location in the subject.
  • Increased clearance of molecules from the CNS of the subject can comprise an increased rate of movement of molecules from the CSF to deep cervical lymph nodes, and thus can be ascertained by monitoring the rate of movement of molecules and/or labels in the CNS to deep cervical lymph nodes.
  • the flow modulator e.g., VEGFR3 agonist and/or FGF2
  • the neurological therapeutic agent is administered selectively to the meningeal space.
  • a composition comprising, consisting of, or consisting essentially of the flow modulator (e.g., VEGR3 agonist and/or FGF2) and/or the neurological therapeutic agent is administered to the CNS, but not outside the CNS.
  • the flow modulator e.g., VEGFR3 agonist and/or FGF2
  • the VEGFR3 agonist can be selected from the group consisting of one or more of the following: VEGF-c, VEGF-d, or an analog, variant, or functional fragment thereof.
  • increasing flow by increasing the diameter of, increasing drainage by, and/or increasing the quantity of meningeal lymphatic vessels as described herein can increase clearance of molecules from the CNS of the subject, and thus reduces the concentration and/or accumulation of the molecules in the CNS and brain in accordance with some embodiments herein. Accordingly, in some embodiments, increasing clearance of molecules in the CNS reduces concentration and/or accumulation of the molecules in the CNS and brain. For example, if amyloid beta plaques are present in the CNS of the subject, increasing clearance can reduce amyloid beta plaques, or decrease the rate of their accumulation.
  • amyloid beta plaques can diminish, or the rate of increase can be reduced.
  • decreases of amyloid beta plaques can represent a decrease in an etiology of a disease caused by amyloid beta plaques, and, more generally can indicate an increase in fluid flow in the CNS, for example via drainage by meningeal lymphatic vessels.
  • a quantity of accumulated amyloid beta plaques in the central nervous system, or the rate of accumulation thereof is reduced by at least 2%, for example at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
  • amyloid beta plaques are cleared from meningeal portions of the central nervous system of the subject.
  • increased fluid flow in the central nervous system of the subject comprises an increased rate of perfusion of fluid throughout an area of the subject’s brain.
  • increased fluid flow in the central nervous system of the subject comprises an increased rate of perfusion out of the subject’s central nervous system.
  • methods, uses, and compositions for increasing clearance of molecules from the CNS can be useful in treating, preventing, or ameliorating symptoms of neurological diseases, for example diseases associated with accumulation of macromolecules, cells, or debris in the CNS. Accordingly, in some embodiments, the method or use further includes determining the subject to have such a neurological disease, or a risk factor for such a neurological disease.
  • Example neurological diseases include AD (such as familial AD and/or sporadic AD), PD, cerebral edema, ALS, PANDAS, meningitis, hemorrhagic stroke, ASD, brain tumor (such as glioblastoma), epilepsy, Down’s syndrome, hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D), Familial Danish/British dementia, dementia with Lewy bodies (DLB), Lewy body (LB) variant of AD, multiple system atrophy (MSA), familial encephalopathy with neuroserpin inclusion bodies (FENIB), frontotemporal dementia (FTD), Huntington’s disease (HD), Kennedy disease/spinobulbar muscular atrophy (SBMA), dentatorubropallidoluysian atrophy (DRPLA); spinocerebellar ataxia (SCA) type I, SCA2, SCA3 (Machado-Joseph disease), SCA6, SCA7,
  • a FGF2 or a VEGFR3 agonist as described herein can be administered.
  • the VEGFR3 agonist is selected from the group consisting of one or more of the following: VEGF-c, VEGF-d, or an analog, variant or functional fragment of either of these, and the neurological therapeutic agent comprises, consists essentially of, or consists of an amyloid beta antibody.
  • the VEGFR3 agonist and/or FGF2 and/or the neurological therapeutic agent is administered selectively to the meningeal space of the subject.
  • the VEGFR3 agonist and/or FGF2 and/or the neurological therapeutic agent e.g., amyloid beta antibody
  • CSF contact cerebral spinal fluid
  • the VEGFR3 agonist and/or FGF2 is administered to the subject after determining the subject to have the risk factor for the neurological disease. In some embodiments, the VEGFR3 agonist and/or FGF2 is administered to the subject after determining the subject to have the neurological disease.
  • the VEGFR3 agonist and/or FGF2 and the neurological therapeutic agent e.g., amyloid beta antibody
  • the present invention is based upon, at least in part, the discovery that the number of immune cells increase in the central nervous system of a subject that has an enhance risk of developing a neurological disease, e.g., Alzheimer’s disease. Accordingly, in some embodiments, the present invention provides a method of reducing the number of immune cells in the central nervous system of a subject.
  • Some embodiments of the disclosure include a method, use, or composition for use in increasing clearance of cells (such as immune cells) from the central nervous system of a subject.
  • the method or use can comprise administering a composition comprising, consisting of, or consisting essentially of a flow modulator (e.g., VEGFR3 agonist and/or FGF2) to a meningeal space of the subject, in which fluid flow in the central nervous system of the subject is increased, and administering a neurological therapeutic agent to the subject.
  • a flow modulator e.g., VEGFR3 agonist and/or FGF2
  • the method or use can increase the clearance of cells from the CNS of the subject.
  • the neurological therapeutic agent may be administered to the meningeal space, or to a different location in the subject.
  • Increased clearance of cells from the CNS of the subject can comprise an increased rate of movement of cells from the CSF to deep cervical lymph nodes, and thus can be ascertained by monitoring the rate of movement of cells and/or labels in the CNS to deep cervical lymph nodes.
  • the flow modulator e.g., VEGFR3 agonist and/or FGF2
  • the neurological therapeutic agent is administered selectively to the meningeal space.
  • a composition comprising, consisting of, or consisting essentially of the flow modulator (e.g. , VEGR3 agonist and/or FGF2) and/or the neurological therapeutic agent is administered to the CNS, but not outside the CNS.
  • the flow modulator e.g., VEGFR3 agonist and/or FGF2
  • the VEGFR3 agonist can be selected from the group consisting of one or more of the following: VEGF-c, VEGF-d, or an analog, variant, or functional fragment thereof.
  • increasing flow by increasing the diameter of, increasing drainage by, and/or increasing the quantity of meningeal lymphatic vessels as described herein can increase clearance of cells, e.g., from the CNS of the subject, and thus reduces the concentration and/or accumulation of the cells, e.g., immune cells, in the CNS and brain in accordance with some embodiments herein. Accordingly, in some embodiments, increasing clearance of cells, e.g., immune cells, in the CNS reduces concentration and/or accumulation of the cells, e.g., immune cells, in the CNS and brain.
  • the flow modulators e.g., VEGF-c
  • the neurological therapeutic agents of the invention synergize with the neurological therapeutic agents of the invention to reduce the number of cells that contribute to the pathogenesis of a neurodegenerative disease, e.g., Alzheimer’s disease, resulting in a greater anti-inflammatory effect than either flow modulator or therapeutic agent alone.
  • Immune cells may contribute to the pathogenesis of a neurodegenerative disease through chronic inflammation.
  • immune cells such as T cells and B cells may contribute to chronic inflammation through secretion of proinflammatory cytokines.
  • proinflammatory cytokines such as Alzheimer’s disease
  • decreases of immune cells can represent a decrease in an etiology of a disease caused by chronic inflammation, and, more generally can indicate an increase in fluid flow in the CNS, for example via drainage by meningeal lymphatic vessels.
  • a quantity of immune cells in the central nervous system, or the rate of accumulation thereof is reduced by at least 2%, for example at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,
  • amyloid beta plaques are cleared from meningeal portions of the central nervous system of the subject.
  • increased fluid flow in the central nervous system of the subject comprises an increased rate of perfusion of fluid throughout an area of the subject’s brain.
  • increased fluid flow in the central nervous system of the subject comprises an increased rate of perfusion out of the subject’s central nervous system.
  • methods, uses, and compositions for increasing clearance of cells, e.g., immune cells, from the CNS can be useful in treating, preventing, or ameliorating symptoms of neurological diseases, for example diseases associated with accumulation of macromolecules, cells, or debris in the CNS. Accordingly, in some embodiments, the method or use further includes determining the subject to have such a neurological disease, or a risk factor for such a neurological disease.
  • Example neurological diseases include AD (such as familial AD and/or sporadic AD), PD, cerebral edema, ALS, PANDAS, meningitis, hemorrhagic stroke, ASD, brain tumor (such as glioblastoma), epilepsy, Down’s syndrome, hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D), Familial Danish/British dementia, dementia with Lewy bodies (DLB), Lewy body (LB) variant of AD, multiple system atrophy (MSA), familial encephalopathy with neuroserpin inclusion bodies (FENIB), frontotemporal dementia (FTD), Huntington’s disease (HD), Kennedy disease/spinobulbar muscular atrophy (SBMA), dentatorubropallidoluysian atrophy (DRPLA); spinocerebellar ataxia (SCA) type I, SCA2, SCA3 (Machado-Joseph disease), SCA6, SCA7,
  • the neurological disease comprises, consists essentially of, or consists of a proteinopathy, for example atauopathy or an amyloidosis such as AD (e.g., familial AD and/or sporadic AD).
  • AD familial AD and/or sporadic AD.
  • a FGF2 or a VEGFR3 agonist as described herein can be administered.
  • the VEGFR3 agonist is selected from the group consisting of one or more of the following: VEGF-c, VEGF-d, or an analog, variant or functional fragment of either of these, and the neurological therapeutic agent comprises, consists essentially of, or consists of an amyloid beta antibody.
  • the VEGFR3 agonist and/or FGF2 and/or the neurological therapeutic agent is administered selectively to the meningeal space of the subject.
  • the VEGFR3 agonist and/or FGF2 and/or the neurological therapeutic agent is administered to the subject by a route selected from the group consisting of at least one of the following: nasal administration, transcranial administration, contact cerebral spinal fluid (CSF) of the subject, pumping into CSF of the subject, implantation into the skull or brain, contacting a thinned skull or skull portion of the subject with the VEGFR3 agonist and/or FGF2 and the neurological therapeutic agent, or expression in the subject of a nucleic acid encoding the VEGFR3 agonist and/or FGF2, or a combination of any of the listed routes.
  • the VEGFR3 agonist and/or FGF2 is administered to the subject after determining the subject to have the risk factor for the neurological disease. In some embodiments, the VEGFR3 agonist and/or FGF2 is administered to the subject after determining the subject to have the neurological disease.
  • the VEGFR3 agonist and/or FGF2 and the neurological therapeutic agent can be administered in an effective amount to treat, inhibit, ameliorate symptoms of, delay the onset of, reduce the likelihood of, prevent the neurological disease.
  • Some aspects provide methods of treating a neurological disease (such as AD) in a subject comprising administering to the subject a therapeutically effective amount of a neurological therapeutic agent and a flow modulator that modulates one or more of a) drainage of the meningeal lymphatic vessel(s); b) diameter of the meningeal lymphatic vessel(s); c) lymphangiogenesis of the meningeal lymphatic vessel(s); d) contractility of the meningeal lymphatic vessel(s); and/or e) permeability of the meningeal lymphatic vessel(s).
  • a neurological disease such as AD
  • the present disclosure also provides methods of treating AD in a subject by administering to the subject a neurological therapeutic agent and a flow modulator that increases drainage of the meningeal lymphatic vessel(s), increases the diameter of the meningeal lymphatic vessel(s), causes lymphangiogenesis of the meningeal lymphatic vessel(s), modulates contractility of the meningeal lymphatic vessel(s) to increase drainage, and/or modulates the permeability of the meningeal lymphatic vessel(s) to increase drainage.
  • the present disclosure also provides methods of treating a neurological disease such as AD described herein in a subject by administering to the subject a neurological therapeutic agent and a flow modulator that increases drainage of the meningeal lymphatic vessel(s), increases the diameter of the meningeal lymphatic vessel(s), causes lymphangiogenesis of the meningeal lymphatic vessel(s), modulates contractility of the meningeal lymphatic vessel(s) to increase drainage, and/or modulates the permeability of the meningeal lymphatic vessel(s) to increase drainage.
  • a neurological disease such as AD described herein in a subject by administering to the subject a neurological therapeutic agent and a flow modulator that increases drainage of the meningeal lymphatic vessel(s), increases the diameter of the meningeal lymphatic vessel(s), causes lymphangiogenesis of the meningeal lymphatic vessel(s), modulates contractility of the meningeal lymphatic vessel(s) to increase drainage, and/or modulates the permeability of the meningeal lymphatic vessel(s) to increase
  • Example 1 Adult 5xFAD meninges and brain
  • FIG. 1A Representative images of the meningeal whole- mounts of 5xFAD mice treated with different combinations of mIgG2a or monoclonal anti-amyloid beta antibody (“ABETA Ab”) with AAVl-CMV-eGFP or AAV 1-CMV-m VEGF-C are shown in FIG. IB.
  • ABETA Ab monoclonal anti-amyloid beta antibody
  • FIGs. ID and 1G the units for the Y-axis are percentage of field of view (“%FOV”). It can be seen that the flow modulator VEGF-c increased lymphatic diameter, and synergized with the amyloid beta antibody to enhance lymphatic branching (FIGs. 1C and IE). These experiments show that a flow modulator and neurological therapeutic agent in accordance with some embodiments herein can synergize to enhance lymphatic branching in the CNS of a model of AD comprising amyloid beta plaques.
  • Example 2 Adult 5xFAD brain
  • b-m Plaque density (number of plaques per mm 2 ), average size (pm 2 ) and coverage (% of brain section) in particular brain regions (cortex and amygdala; hippocampus; thalamus and hypothalamus) or in the whole brain section.
  • amyloid plaque size and brain coverage were reduced in several regions of mice receiving combination treatment of AAV1 expressing VEGF-c and antibody, particularly when, compared to mice receiving control mIgG2a only (see FIGs. 2F-2M).
  • This experiment shows that a flow modulator and neurological therapeutic agent in a model of AD comprising amyloid beta plaques in accordance with some embodiments herein can synergize to reduce amyloid beta plaque size, density, and coverage (as percent of brain region).
  • FIG. 3A Age of APPswe mice and treatment regimen of anti -amyloid beta antibody with AA1 vector expressing VEGF-c or eGFB via i.c.m are shown in FIG. 3A.
  • Results of behavioral tests (open field (OF), novel location recognition (NLR) and contextual fear conditioning (CFC)) are shown in FIGs 3B-3D.
  • Administration of VEGF-c treatment reduced the number of plaques in the cortex and amygdala (FIG. 3F) or in the hippocampus (FIG. 3G) Total distance, velocity and time in center of the arena (% of total time) in the OF test are shown in FIG. 3B.
  • Time investigating one of the object location (% of total time investigating objects) in the training trial and time investigating the novel object location (% of total time investigating) in the NLR test are shown in FIG. 3C.
  • Time freezing (% of total time) in the context trial and in cued trial of the CFC are shown in FIG. 3D.
  • FIG. 3E Representative images of the brain sections of APPswe mice treated with anti-Abeta antibody and with AAVl-CMV-eGFP or AAVl-CMV-mVEGF-C are shown in FIG. 3E. Brain sections were stained for Ab and with DAPI; scale bar, 1mm.
  • Example 4 Alzheimer’s Disease and Introduction
  • AD Alzheimer’s disease
  • a growing body of evidence points to passive immunotherapy as a promising therapeutic strategy to slow AD progression.
  • Administration of monoclonal antibodies against amyloid beta (Ab) has been shown to reduce brain senile plaque load both in AD transgenic mouse models and in AD patients. While dysfunctional meningeal lymphatic vasculature plays an important role in Ab accumulation, it was previously unknown if or how altered brain drainage mediated by meningeal lymphatics affects immunotherapy in AD.
  • AD Alzheimer's disease
  • meningeal lymphatic dysfunction exacerbates brain and meningeal Ab pathology in different transgenic mouse models of familial AD (Da Mesquita, S. et al. Functional aspects of meningeal lymphatics in ageing and Alzheimer's disease. Nature 560, 185-191, (2016)). Accordingly, it was hypothesized that altered meningeal lymphatic function would affect brain fluid drainage and recirculation, thereby changing the availability of monoclonal antibodies administered to target and clear brain Ab deposits. This hypothesis led to further investigate changes in the meningeal lymphatic vasculature at different ages in transgenic mouse models of AD, as well as the potential therapeutic implications of modulating this brain- draining meningeal lymphatic system.
  • RNA-seq meningeal lymphatic endothelial cells
  • FIG. 5F and FIGs. 6A-6C meningeal lymphatic endothelial cells
  • Tables 2-7 summarize the results as shown in FIGs. 6A-6C. It was found that at this relatively young age, LECs taken from the 5xFAD mice already showed significant changes in the expression of genes involved in the regulation of Golgi apparatus and exocytosis, phospholipase D signaling, and response to low-density lipoprotein (FIGs. 5G-5I and FIGs. 6B and 6C). Tables 8 and 9 summarize the results as shown in FIG. 5H.
  • FIG. 7A-7D revealed a significant increase in the numbers of B cells, CD4 + T cells and CD8 + T cells in middle-aged (11-12 months-old) mice relative to their numbers at 5-6 months (FIG. 7E).
  • Table 18 summarizes the results as shown in FIG. 7B.
  • the degeneration of lymphatic vasculature observed in middle-aged 5xFAD mice was corroborated by an accumulation of adaptive immune cells in the meninges, which — as shown in a previous study (Louveau, A. etal. CNS lymphatic drainage and neuroinflammation are regulated by meningeal lymphatic vasculature. Nat Neurosci 21, 1380-1391, (2016)) — can be indicative of impaired meningeal lymphatic drainage.
  • Example 6 Meningeal lymphatic drainage affects anti-Ab immunotherapy [0430]
  • incubation of brain sections from WT or 5xFAD mice confirmed that anti-Abeta antibody specifically recognizes human Ab deposited in the brains of 5xFAD mice, but not of WT mice (FIG. 8A).
  • Murine IgG2a isotype antibody (mIgG2a, clone 4-4-20e), used here as a control, did not recognize Ab plaques in brain sections from 5xFAD mice (FIG. 8A).
  • anti-Abeta antibody was injected into 5-month-old 5xFAD mice, either via the CSF (5 pL at 1 pg/pL) by intra-cistema magna (i.c.m.) infusion or intravenously (i.v., 100 pL at 1 pg/pL). Both at 1 h and at 24 h after anti-Abeta antibody injection, the recognition of Ab plaques was more pronounced when anti-Abeta antibody was administered into the CSF (FIG. 8B) than via the i.v. route (FIG. 8C). The results, in agreement with a recent report (Plog, B. A. et al.
  • CNS lymphatic drainage and neuroinflammation are regulated by meningeal lymphatic vasculature. Nat Neurosci 21, 1380-1391, (2016); Louveau, A. et al. Structural and functional features of central nervous system lymphatic vessels. Nature 523, 337-341, (2015)), namely along the transverse sinus, for drainage of CSF components into the dCLNs, and call into question a more recent assertion specifying a key role for the lymphatics in the basal region of the meninges (Ahn, J. H. et al. Meningeal lymphatic vessels at the skull base drain cerebrospinal fluid. Nature 572, 62-66, (2019)).
  • mice with intact meningeal lymphatic vasculature presented significantly less Ab plaque load than mice with ablated meningeal lymphatics across different brain regions, especially in the cortex (FIGs. 8D-8G and FIGs. 8M and 8N).
  • FIGs. 11A-11G the heightened neuroinflammatory response observed in 5xFAD mice with ablated meningeal lymphatic vessels was concurrent with behavioral deficits (FIGs. 11A-11G).
  • Mice with impaired meningeal lymphatics spent less time in the center of the open field arena (FIGs. 11A- 11D) and took more time to find the submerged platform in the acquisition of the Morris water maze test (FIGs. 11E-11G), when compared to their control counterparts.
  • Prolonged treatment with the monoclonal antibody ABETA Mab 1 was not able to improve the performance of 5xFAD mice in the open field or the Morris water maze (FIGs. 11A-11G).
  • Brain myeloid phagocytes namely microglia and recruited macrophages, were shown to be closely involved in Fc receptor-mediated clearance of parenchymal Ab aggregates upon injection of monoclonal antibodies against Ab (Bard, F. etal. Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease. Nat Med 6, 916-919, (2000); Wilcock, D. M. etal. Microglial activation facilitates Abeta plaque removal following intracranial anti-Abeta antibody administration. Neurobiol Dis 15,
  • FIG. 13D A Unique Microglia Type Associated with Restricting Development of Alzheimer's Disease. Cell 169, 1276-1290 el217, (2017)) (FIGs. 13C and 13D). Table 19 summarizes the results as shown in FIG. 13D.
  • analysis of the genes that are differentially expressed in the two groups revealed a downregulation of hexosaminidase subunit beta ( Hexb ) and an upregulation of apolipoprotein E ⁇ Apoe) across all microglial clusters, and these changes were significant when all cells were pooled by group (FIGs. 13E-13H).
  • Example 7 Enhancing meningeal lymphatics modulates anti-Ab immunotherapy
  • Most monoclonal anti-Ab antibodies tested in clinical trials have failed to significantly prevent cognitive decline in AD patients, possibly owing to serious side effects such as deleterious activation of blood vasculature and microglia (Ryu, J. K. etal. Fibrin-targeting immunotherapy protects against neuroinflammation and neurodegeneration. Nat Immunol 19, 1212-1223, (2016); Merlini, M. et al. Fibrinogen Induces Microglia-Mediated Spine Elimination and Cognitive Impairment in an Alzheimer's Disease Model. Neuron 101, 1099-1108 el096, (2019); Hong, S. et al.
  • VEGF-C murine vascular endothelial growth factor- C
  • FIG. 15A adeno-associated virus 1 (AAVl)-mediated expression of murine vascular endothelial growth factor- C
  • AAVl adeno-associated virus 1
  • mVEGF-C murine vascular endothelial growth factor- C
  • FIG. 15A Increased VEGF-C signaling through VEGFR3 expressed by meningeal LECs was previously shown by research groups to improve meningeal lymphatic function and CSF drainage
  • mice treated with mVEGF-C and anti-Abeta antibody (ABETA Mabl)
  • measurements of brain Ab plaque load, neurite dystrophy (assessed by the levels of lysosomal -associated membrane protein 1), vascular fibrinogen deposition, and myeloid (IBA1 + ) cell response at the end point of the treatment regimen revealed a significant reduction of Ab plaque coverage throughout the entire forebrain (FIGs. 15N-15R), less cortical vascular fibrinogen (FIGs. 15S-15U), fewer IBA1 + cells clustering around Ab deposits, and decreased amounts of CD68 in IBA1 + cells (FIGs.
  • FIG. 16F Representative images of the brain sections of J20 mice treated with ABETA Mabl and with the antibody and either AAVl-CMV-eGFP or AAV 1-CMV-m VEGF-C are shown in FIG. 16F. Brain sections were stained for Ab and with DAPI.
  • FIGs. 16G-16I show amyloid beta plaque coverage (% of brain section) in the hippocampus, cortex and amygdala, or in the hippocampus, cortex and amygdala combined.
  • Amyloid beta plaque coverage in the hippocampus, cortex and amygdala were lessened in mice that received AAV 1 -vector expressing VEGF-c (see FIGs. 16F-16I).
  • This experiment shows that a flow modulator and neurological therapeutic agent in an additional model of AD (J20 mice) comprising amyloid beta plaques in accordance with some embodiments herein can synergize to reduce amyloid beta plaque coverage (as percent of brain region).
  • Example 8 Disease-associated genes are highly expressed by lymphatic endothelial cells [0443] To further explore the relationship between changes in the lymphatic system and AD, it was hypothesized that higher risk for AD, owing to disease-associated single nucleotide polymorphisms (SNPs), could be directly linked to altered function of the lymphatic vasculature. During the last decade, genome-wide association studies have revealed numerous genes containing SNPs that affect the risk for AD (Guerreiro, R. el al. TREM2 variants in Alzheimer's disease. N Engl JMed 368, 117- 127, (2013); Naj, A. C. et al.
  • Table 28 summarizes the results as shown in FIG. 18D.
  • Functional pathway analysis using disease-associated genes expressed in LECs revealed changes in vascular development (G0:0001944), cell projection (G0:0048858) and tube morphogenesis (G0:0030198), negative regulation of cell differentiation (G0:0045596) and migration (G0:0030334) and positive regulation of cell death (G0:0010942) across two or more diseases (FIG. 17C).
  • AD-related pathways changes were also observed in cell-matrix adhesion (G0:0007160), cell-cell junction (G0:0005911), amyloid beta metabolic process (G0:0050435) and lipoprotein particle binding (G0:0071813), which stress out once again that altered gene expression in LECs might lead to structural and morphological changes in the lymphatic vasculature and potentially impact on lymphatic vessel integrity, LEC survival and Ab metabolism (FIG. 17C).
  • meningeal LECs and brain BECs presented a higher proportion of highly expressed AD-associated genes (FIGs 17A, 17B, 17C and FIGs. 18C, 18D), when compared to microglia from the 5xFAD cortex (FIGs. 18E-18G).
  • meningeal lymphatic endothelial cells have signatures that distinguish them from diaphragm and skin lymphatics.
  • Gene expression profiles were also evaluated in meningeal lymphatics of young and old mice. Changes in genes associated with immune system, growth factor, and extracellular matrix pathways in meningeal lymphatics were observed (FIG. 24B).
  • results of combination therapy with mVEGF-C either prophylactic to prevent meningeal lymphatic dysfunction in 4-5 months-old 5xFAD mice, or therapeutic to augment meningeal lymphatic drainage in aged J20 and APPswe mice (both of which develop less brain Ab pathology and at a later age), show synergistically improved clearance of Ab by anti-Abeta antibody.
  • results also seem to indicate that administering anti-Ab antibody together with mVEGF-C directly into the CSF of aged mice, in detriment of a peripheral route of administration that has to rely on transport mechanisms across the blood-brain barrier, might improve Ab plaque clearance from the brain.
  • APOE disease-related proteins
  • Tau Y anamandra, K. et al. Anti-tau antibodies that block tau aggregate seeding in vitro markedly decrease pathology and improve cognition in vivo. Neuron 80, 402-414, (2013)
  • fibrin Rost J. K. et al. Fibrin-targeting immunotherapy protects against neuroinflamm
  • meningeal lymphatic vasculature might be of relevance for other neurodegenerative disorders characterized by protein misfolding and accumulation, such as Huntington’s or Parkinson’s diseases, where administration of antisense oligonucleotides (Kordasiewicz, H. B. et al. Sustained therapeutic reversal of Huntington's disease by transient repression of huntingtin synthesis.
  • mice In-house bred male or female transgene carriers and non-carrier (WT) littermates were used at different ages. The genotype and age of mice from different strains are indicated in figure schemes or legends throughout the manuscript. Male mice were used in the different experiments, unless stated otherwise. Mice of all strains were housed in an environment with controlled temperature and humidity and on a 12-hour light/dark cycle (lights on at 7:00). All mice were fed with regular rodent’s chow and sterilized tap water ad libitum. All experiments were approved by the Institutional Animal Care and Use Committee of the University of Virginia.
  • ABETA Mabl and respective control IgG2a (mlgG) antibodies were manufactured by Absolute Antibody Utd., Oxford Centre for Innovation, United Kingdom.
  • Anti-Ab monoclonal antibodies and respective mlgG controls were administered via intraperitoneal (i.p.) injection, at a dose of 40 mg/kg.
  • Antibody dosages are also specified in the main text, illustrated in schemes and in each figure legend.
  • ABETA Mabl and mlgG antibodies were injected directly into the CSF, following the methodology described in the next section.
  • the volume of the desired solution was injected into the CSF-fdled cistema magna compartment at a rate of ⁇ 2.5 pL/min. After injecting, the syringe was left in place for at least 2 min to prevent back-flow of CSF. The neck skin was then sutured, the mice were allowed to recover in supine position on a heating pad until fully awake and subcutaneously injected with ketoprofen (2 mg/Kg).
  • This method of intra-cistema magna (i.c.m.) injection was used to administer 5 pF of either Visudyne® (verteporfm for injection, Valeant Ophtalmics), adeno associated viral vectors (AAV1- CMV-mVEGF-C-WPRE and AAVl-CMV-eGFP at 10 12 genome copies per mF, purchased from Vector BioFabs, Philadelphia) or different antibody solutions of murine Abeta MAb or IgG2a (manufactured by Absolute Antibody Ftd., Oxford Centre for Innovation, United Kingdom).
  • antibodies were also injected into the tail vein of mice (i.v.). Antibody dosages/titers are specified in the main text and in each figure legend.
  • Meningeal lymphatic vessel ablation Selective ablation of the meningeal lymphatic vessels was achieved by injection of Visudyne (Vis.) and consecutive transcranial photoconversion (photo.) steps following previously described methodology and regimens (Da Mesquita, S. etal. Functional aspects of meningeal lymphatics in ageing and Alzheimer's disease. Nature 560, 185-191, (2016); Fouveau, A. etal. CNS lymphatic drainage and neuroinflammation are regulated by meningeal lymphatic vasculature. Nat Neurosci 21, 1380-1391, (2016)). Visudyne was reconstituted following manufacturer instructions, aliquoted and kept at -20°C until further used.
  • Visudyne was injected into the CSF (i.c.m.) and, 15 min later, an incision was performed in the skin to expose the skull bone and photoconvert the drug by pointing a 689-nm wavelength non- thermal red light (Coherent Opal Photoactivator, Lumenis) on 5 different spots above the intact skull (close to the injection site, above the superior sagittal sinus close to the rostral rhinal vein, above the confluence of sinuses and above each transverse sinus). Each spot was irradiated with a light dose of 50 J/cm 2 at an intensity of 600 mW/cm 2 for a total of 83 s.
  • a 689-nm wavelength non- thermal red light Coherent Opal Photoactivator, Lumenis
  • Controls were injected with the same volume ofVisudyne only, without the photoconversion step.
  • the scalp skin was then sutured, the mice were allowed to recover on a heating pad until fully awake and subcutaneously injected with ketoprofen (2 mg/Kg).
  • the mouse was positioned supine with the head held in position with a length of suture behind the upper incisors and the upper limbs held in place with medical tape. Incisions were made from the center of the clavicle, anterior to the top of the salivary gland and lateral approximately 1 cm. The further preparation was performed on the right side, however in some instances moved to the left side when anatomical variation prevented imaging. The salivary gland was carefully separated at its lateral extent and gently retracted medially. The omohyoid and stemomastoid muscles were retracted laterally, exposing the dCLN. Imaging began approximately 15 minutes after i.c.m. injection. Images were acquired at 25-30 frames per second for a total of 60 seconds.
  • mice were euthanized by injection of Euthasol (10% v/v in saline). Fluorescent microsphere drainage was analyzed in FIJI software by drawing a line demarcating the draining lymphatic vessel afferent to the dCLN and manually counting the beads passing the line by a blinded experimenter. Mice were discarded from the analysis due to prior complications during the surgical procedure (e.g. hemorrhages) or due to failure in detecting microspheres draining into the dCLN during image acquisition. In representative images, microspheres were tracked using TrackMate (Tinevez, J. Y. etal. TrackMate: An open and extensible platform for single-particle tracking. Methods 115, 80-90, (2017)) to show the comulative tracks over a 20 sec interval.
  • mice were habituated to the behavior room, including the background white noise, for at least 30 min before starting the test.
  • the MWM test consisted of 4 days of acquisition, 1 day of probe trial and 2 days of reversal. In the acquisition, mice performed four trials per day, for 4 consecutive days, to find a hidden 10-cm diameter platform located 1 cm below the water surface in a pool 1 m in diameter.
  • Tap water was made opaque with nontoxic white paint (Prang ready -to-use washable tempera paint) and the water temperature was kept at 25 ⁇ 1°C by addition of warm water.
  • a dim light source was placed within the testing room and only distal visual cues were available above each quadrant of the swimming pool to aid in the spatial navigation and location of the submerged platform.
  • the latency to platform i.e., the time required by the mouse to find and climb onto the platform, was recorded for up to 60 seconds. Each mouse was allowed to remain on the platform for ⁇ 15 seconds and was then moved from the maze to its home cage. If the mouse did not find the platform within 60 seconds, it was manually placed on the platform and returned to its home cage after ⁇ 15 seconds.
  • the inter-trial interval for each mouse was of at least 5 min. On day 5, the platform was removed from the pool, and each mouse was tested in a probe trial for 60 seconds. On days 1 and 2 of the reversal, without changing the position of the visual cues, the platform was placed in the quadrant opposite to the original acquisition quadrant and the mouse was retrained for four trials per day.
  • mice were given a lethal dose of anesthetics by intraperitoneal (i.p.) injection of Euthasol (10% v/v in saline) and transcardially perfused with ice cold phosphate buffer saline (PBS, pH 7.4) with heparin (10 U/mL). After stripping the skin and muscle from the bone, the entire head was collected and drop fixed in 4% paraformaldehyde (PFA) for 24 hours at 4°C.
  • PFA paraformaldehyde
  • the top of the skull was removed with fine surgical curved scissors (Fine Science Tools) by cutting clockwise, beginning and ending inferior to the right post-tympanic hook and kept in PBS 0.02% azide at 4°C until further use.
  • Fine surgical curved scissors Fine Science Tools
  • Fixed meninges were carefully dissected from the skullcaps with Dumont #5 and #7 fine forceps (Fine Science Tools) and kept in PBS 0.02 % azide at 4°C until further use.
  • the skull was cut sagitally, along the median plane, and after removing the brain, the skull pieces with the attached meningeal layers were kept in PBS 0.02 % azide at 4°C until further use.
  • the brains were kept in 4% PFA for additional 24 hours (48 hours in total).
  • Fixed brains were washed with PBS, cryoprotected with 30% sucrose and frozen in Tissue-Plus ® O.C.T. compound (Thermo Fisher Scientific). Fixed and frozen brains were sliced (50 pm thick sections) with a cryostat (Feica) and kept in PBS 0.02% azide at 4°C.
  • rat anti-LYVE-l-eFluor660 or anti-LYVE-l-Alexa Fluor ® 488 eBioscience, clone ALY7, 1:200
  • Armenian hamster anti-CD31 Millipore Sigma, MAB1398Z, clone 2H8, 1:200
  • rabbit anti-AQP4 Millipore Sigma, A5971, 1:200
  • goat anti-IBAl Abeam, ab5076, polyclonal, 1:200
  • rat anti-CD68 BioLegend, 137002, clone FA-11, 1:100
  • rat anti -LAMP- 1 Abeam, ab25245, clone 1D4B, 1:300
  • rabbit anti-Fibrinogen Dako, A0080, polyclonal, 1:200
  • anti -Ab 1-37/42 Cell Signaling, 8243S, clone D54D2, 1:
  • Meningeal whole mounts or brain sections were then washed 3 times for 10 min at RT in PBS-T followed by incubation with the appropriate rat, chicken, goat or donkey eFluor570 or Alexa Fluor ® 488, 594, or 647 conjugated anti-rat, -goat, -rabbit, -mouse or -Armenian hamster IgG antibodies (Thermo Fisher Scientific, 1:500) for 1 hour at RT in PBS-T.
  • Skull caps were washed once with PBS kept in PBS at 4°C. Preparations were stored at 4°C for no more than 1 week until images were acquired.
  • a stereomicroscope M205 FA, Leica Microsystems was used to image the meningeal lymphatic vessels within the skull caps.
  • a widefield microscope (DMI6000 B with Adaptive Focus Control, Leica Microsystems) was used for images of Ab deposits in brain sections and a confocal microscope (FV1200 Laser Scanning Confocal Microscope, Olympus) to acquire all the other images. Upon acquisition, images were opened in the FIJI software for quantification.
  • the ROI (region of interest) manager, Simple Neurite Tracer and Cell Counter plugins were used to measure total lymphatic vessel length and branching points in a particular region of the meningeal whole mount.
  • the Threshold and Measure plugins were used to measure the coverage (as % of ROI or as area in pm 2 ) by Ab in the brain (in delineated hippocampus, cortex/striatum/amygdala, thalamus/hypothalamus, or whole brain section; plotted values resulted from the average of 3 representative sections per sample) and meninges, as well as LAMP-1, Fibrinogen and IBA1 in images of the brain cortex (plotted values resulted from the average of 4 representative images taken from 2 brain sections per sample).
  • the Analyze Particles plugin (Size, 4- infmity pm 2 ; Circularity, 0.05-1) was used to measure the number of Ab plaques per mm 2 of brain region/section and average size of the plaques (pm 2 ).
  • the Cell Counter plugin was also used to quantify the number of peri-Ab plaque IBA1 + cells (cell body within 10 pm of plaque).
  • the Threshold and Image Calculator plugins were used to determine the % of colocalization between the signals of anti-Abeta antibody and CD31, anti-Abeta antibody and Ab (Amylo-Glo RTD), or IBA1 and CD68 in brain images acquired using the confocal microscope. All measurements were performed by a blinded experimenter, Microsoft Excel was used to calculate average values in each experiment and statistical analysis performed using Prism 7.0a (GraphPad Software, Inc.).
  • the tissues were digested for 20 min at 37°C with 1 mg/mL of Collagenase VIII, 1 mg/mL of Collagenase D and 50 U/mL of DNAse I (all from Sigma Aldrich) in RPMI 1640.
  • FACS fluorescence-activated cell sorting
  • Cell viability was determined by using the Zombie NIRTM or Zombie AQUATM Viability Kits following the manufacturer’s instructions (BioLegend). After an incubation period of 25 min at 4°C, cells were washed with FACS buffer and fluorescence data was collected with a GalliosTM Flow Cytometer (Beckman Coulter, Inc.). Data was analyzed using FlowJoTM 10 software (Tree Star, Inc.). Briefly, singlets were gated using the height, area and the pulse width of the forward and side scatters and then viable leukocytes were selected as CD45 + Zombie NIR neg or Zombie AQUA neg (CD45 + live). Cells were then gated for the appropriate cell type markers.
  • mice were euthanized and transcardially perfused, skull caps were collected, and brain meninges were harvested and digested to obtain a final single cell suspension following the same methodology described in the flow cytometry section.
  • Cell suspensions resulting from each brain meninges were transferred in a 96- well plate and washed with MP PBS.
  • An aliquot from the unstained single cell suspensions was incubated with ViaStainTM AOPI Staining Solution to provide accurate counts for each sample using Cellometer Auto 2000. Unless stated otherwise, all washes and incubations were performed with Maxpar Cell Staining Buffer (MP CSB).
  • MP CSB Maxpar Cell Staining Buffer
  • the fixed cells were washed and stained for intracellular antigens in MP Nuclear Antigen Staining Buffer Set (Fluidigm) for additional 30 min at RT with: anti -TNF-Pr- 141 (clone MP6-XT22, Fluidigm 3141013B), anti-Foxp3-Gd-158 (clone FJK-16s, Fluidigm 3158003A) anti-RORgt-Tb-159 (clone B2D, Fluidigm 3159019B), anti-T-bet-Ho-165 (clone 4B10, Maxpar Ready, conjugated in- house) and anti-GATA3-Er-167 (clone TWAJ, Fluidigm 3167007A).
  • MP Nuclear Antigen Staining Buffer Set Frluidigm
  • a subset of cells was selected for t-distributed stochastic neighbor embedding (tSNE) visualization by randomly sampling an equal number of cells from each replicate, totaling 12,000 cells from each condition. Frequencies were calculated as the number of live CD45 + cells from each sample belonging to each cluster divided by the total number of live CD45 + cells in that sample. Frequencies were then used to calculate the number of cells in each cluster for each group.
  • tSNE stochastic neighbor embedding
  • Suspensions of meningeal LECs were pelleted, resuspended in ice-cold FACS buffer containing DAPI (1: 1000, Thermo Fisher Scientific), anti- CD45-BB515 (1:200, clone 30-F11, BD Biosciences), anti-CD31-Alexa Fluor ® 647 (1:200, clone 390, BD Biosciences) and anti-Podoplanin-PE (1:200, clone 8.1.1, eBioscience) and incubated for 15 min at 4°C. Cells were then washed and resuspended in ice-cold FACS buffer. Briefly, singlets were gated using the pulse width of the side scatter and forward scatter.
  • RNA samples were stored at -80°C until further use.
  • Hippocampus dissection and RNA isolation This procedure was performed as previously described, with minor modifications.
  • hippocampus was macrodissected from the right brain hemisphere in ice-cold advanced DMEM/F12 (Gibco, 12634010) using Dumont #5 and #7 fine forceps and immediately snap-frozen in dry ice and stored at -80 °C until further use. After defrosting on ice, samples were mechanically dissociated in the appropriate volume of extraction buffer from the RNA isolation kit (RNeasy mini kit, 74106, QIAGEN). Total RNA from each sample was isolated and purified using the kit components according to the manufacturer’s instructions.
  • RNA sequencing The Illumina TruSeq Stranded Total RNA Library Prep Kit was used for cDNA library preparation from total RNA samples isolated from cultured human dermal lymphatic microvascular endothelial cells. Sample quality control was performed on an Agilent 4200 TapeStation Instrument, using the Agilent D1000 kit, and on the Qubit Fluorometer (Thermo Fisher Scientific). For RNA sequencing (RNA-seq), libraries were loaded on to aNextSeq 500 (Illumina) using Illumina NextSeq High Output (150 cycle, #FC-404-2002) and Mid Output (150 cycle, #FC- 404-2001) cartridges.
  • RNA-seq RNA sequencing
  • RNA-seq Processing of total RNA extracted from mouse meningeal LECs (including linear RNA amplification and cDNA library generation) and RNA-seq was performed by HudsonAlpha Genomic Services Laboratory (Huntsville, AL).
  • the fastq files were downloaded using HudsonAlpha’ s provided software and fastq files were merged by lane.
  • the merged fastq files were then chastity filtered to remove low quality bases, trimmed using the trimmomatic software, and mapped to the UCSC mm 10 genome using Salmon (Harrow, J. el al. GENCODE: the reference human genome annotation for The ENCODE Project. Genome Res 22, 1760-1774, (2012); Patro, R., Duggal, G., Love, M. I., Irizarry, R. A. & Kingsford,
  • RNA-seq data of LECs from diaphragm or from ear skin were included in the heatmap visualizations for reference but were not used for calculation of the average expression. Heatmaps were visualized using the log2 transformed expression values and the pheatmap package. Genes falling within the top 25 th percentile of highly expressed genes across the LEC datasets were used as gene sets for functional enrichment of GO terms and KEGG pathways using Fischer’s exact test as implemented in the ClusterProfiler package(Yu, G., Wang, L. G., Han, Y. & He, Q. Y. clusterProfiler: an R package for comparing biological themes among gene clusters.
  • the average normalized expression of each gene was calculated within each cluster and the percentage of disease-associated genes falling within each quantile was determined based on average expression of the gene within the cluster.
  • the union of the sets of disease-associated genes falling within the top 2 nd percentile of highly expressed genes for each cluster was visualized in the heatmap using log2 average normalized expression values.
  • mice Brain myeloid cell sorting and single-cell RNA sequencing.
  • 5xFAD mice were injected (i.c.m.) with Visudyne alone (3 mice) or with Visudyne followed by transcranial photoconversion steps (3 mice) following previously described methodology. The same procedures were repeated three weeks later.
  • mice were euthanized by i.p. injection of Euthasol and transcardially perfused with ice cold PBS with heparin. The skulls were collected, and the brain was carefully extracted into ice-cold HBSS medium (14025, Thermo Fisher Scientific).
  • Brain tissue was passed through a 5 mL pipette tip (10 times), and digested for 30 min at 37°C in HBSS with 1 mg/mL of Collagenase VIII, 1 mg/mL of Collagenase D, 50 U/mL of DNAse I and 25 pg/mL Actinomycin D to inhibit transcription mediated by all RNA polymerases (all from Sigma Aldrich).
  • the cell pellets were washed, resuspended in ice-cold FACS buffer, preincubated for 10 min at 4°C with Fc- receptor blocking solution (rat anti-mouse CD16/32, clone 93, BioLegend, 1:200 in FACS) and stained for extracellular markers with the following antibodies (all at 1:200 in FACS): anti-CD 1 lb PerCP-Cy5.5 (550993, BD Biosciences), anti-CD45 A700 (560510, BD Biosciences) and anti-Ly6G BV421 (562737, BD Biosciences).
  • Fc- receptor blocking solution rat anti-mouse CD16/32, clone 93, BioLegend, 1:200 in FACS
  • Single myeloid cells from samples pertaining to the same group were pooled into the same 1.5 mL tubes containing 0.04% non-acetylated BSA in PBS and diluted to 1000 cells per pL estimated from counting on a hemocytometer and Trypan blue (Sigma Aldrich) staining.
  • the sorted myeloid cells (-2000 per sample) were loaded onto a ChromiumTM Single Cell A Chip (PN-120236, 10X Genomics) placed in a 10XTM Chip Holder and run on a 10XTM platform ChromiumTM Controller to generate cDNAs carrying cell- and transcript- specific barcodes. Sequencing libraries were constructed using the ChromiumTM Single Cell 3’ Library & Gel Bead Kit v2 (PN-120237, 10X Genomics).
  • tissue suspensions were sequentially passed through a 10 mL serological pipette (10 times), followed by two passages through a 5 mL serological pipette (10 times each).
  • Cellular suspensions were filtered through a 70 pm cell strainer, thoroughly mixed with an equal volume of 22% BSA in PBS and centrifuged at 1,000 x g for 10 min at RT (without break), to remove the myelin.
  • the cell pellets were washed with advanced DMEM/F12 supplemented with 10 % FBS, resuspended in ice-cold FACS buffer, preincubated for 10 min at 4°C with Fc-receptor blocking solution (anti-CD 16/32, 101302, BioLegend, 1:200 in ice-cold FACS buffer) and stained for extracellular markers with the following antibodies (at 1:200 in ice-cold FACS buffer, unless stated otherwise): anti-CD 13-FITC (at 1:50, 558744, BD Biosciences), anti- Ly6G-PE (127608, BioLegend), anti-CDllb PerCP-Cy5.5 (550993, BD Biosciences), anti-CD31- APC (17-0311-80, eBioscience) and anti-CD45-APC-Cy7 (103116, BioLegend).
  • Fc-receptor blocking solution anti-CD 16/32, 101302, BioLegend, 1:200 in ice-col
  • DAPI 0.1 pg/mL was also added to access cell viability. After an incubation period of 25 min at 4°C, cells were washed and resuspended in ice-cold FACS buffer. Cells were gated on DAPF singlets (based on height, area and the pulse width of the forward and side scatters), CD45 + CD1 lb + Ly6G (myeloid cells), CD45 CD1 lb CD13 + CD31 (mural cells) and CD45 D1 lb CD31 + (blood endothelial cells) and sorted using the InfluxTM Cell Sorter at the University of Virginia Flow Cytometry Core Facility.
  • the aforementioned enriched single cell populations pertaining to the same group were sorted into the same 1.5 mL tube previously coated (overnight) with 0.1% ultrapure non-acetylated BSA (Thermo Fisher Scientific, AM2616) in PBS and containing 500 pL of ice-cold advanced DMEM/F12. Single cells were centrifuged at 350 c g for 5 min at 4°C and resuspended in 0.1% ultrapure non-acetylated BSA in PBS at 1,000 cells per pL estimated from counting on a hemocytometer and Trypan blue (MilliporeSigma) staining. Single-cell suspensions were kept on ice until further use.
  • Brain tissue was passed through a 5 mL pipette tip (10 times) and digested for 30 min at 37°C in HBSS with 1 mg/mL of Collagenase VIII, 1 mg/mL of Collagenase D, 50 U/mL of DNAse I and 25 pg/mL Actinomycin D to inhibit transcription mediated by all RNA polymerases (all from MilliporeSigma).
  • the same volume of DMEM/F12 (Gibco) with 5% FBS and 10 mM EDTA was added to the digested tissue, which was then passed through a 1 mL pipette tip (10 times) and filtered through a 70 pm cell strainer.
  • the cell pellets were washed, resuspended in ice-cold FACS buffer, preincubated for 10 min at 4°C with Fc-receptor blocking solution (anti-CD 16/32, 1:200 in FACS buffer) and stained for extracellular markers with the following antibodies (all at 1:200 in FACS buffer): anti-CD Hb-PerCP-Cy5.5 (550993, BD Biosciences), anti-CD45-A700 (560510, BD Biosciences) and anti-Ly6G-BV421 (562737, BD Biosciences).
  • Fc-receptor blocking solution anti-CD 16/32, 1:200 in FACS buffer
  • Single myeloid cells from samples pertaining to the same group were sorted into the same 1.5 mL tubes previously coated with and containing 500 pL of ice-cold 0.1% ultrapure non-acetylated BSA in PBS. Single cells were centrifuged at 350 c g for 5 min at 4°C and resuspended in the same buffer to 1,000 cells per pL estimated from counting on a hemocytometer and Trypan blue (Millipore Sigma) staining. Single-cell suspensions were kept on ice until further use.
  • Murine single-cell RNA sequencing For the experiments involving sorted blood vascular and myeloid cells from whole brain hemispheres, -10,000 cells per sample were loaded onto a ChromiumTM Single Cell A Chip (PN-120236, lOx Genomics) placed in a lOxTM Chip Holder and run on a lOxTM platform ChromiumTM Controller to generate cDNAs carrying cell- and transcript-specific barcodes. For the experiment involving sorted myeloid cells only from the brain cortex, -2,000 cells per sample were loaded onto the ChromiumTM Single Cell A Chip and run on a lOxTM platform ChromiumTM Controller to generate the cDNAs.
  • ChromiumTM Single Cell A Chip PN-120236, lOx Genomics
  • ChromiumTM Controller ChromiumTM Controller
  • Sequencing libraries were constructed using the ChromiumTM Single Cell 3’ Library & Gel Bead Kit v2 (PN-120237, lOx Genomics). After a quality control (QC) step performed on an Illumina MiSeqNano system, libraries were sequenced on the Illumina NextSeq platform using paired-end sequencing, with 100,000 reads targeted per cell. All murine single-cell RNA-seq data were generated in the Genome Analysis and Technology Core of the University of Virginia (RRID:SCR_018883). Binary base call (bcl) files were converted to fastq format using the Illumina bcl2fastq2 software. Fastq files were then mapped to the mm 10 transcriptome using the Cellranger 3.0.2 pipeline, specifically the count function.
  • the resulting gene by count matrices were then read into R and filtered to remove low quality cells based on unique molecular counts, unique genes, and percent mitochondrial gene expression. Additionally, genes were filtered to retain only those which were expressed more than five cells. The remaining cells were then normalized, and log transformed using the scran normalization package. The resulting normalized counts were then transformed from log2 scale to the natural log scale for compatibility with Seurat v3 and the effects of sequencing depth per cell, number of unique features, and percentage of mitochondrial genes were regressed out. Highly variable genes were determined using the variance stabilizing transformation and the top 2,000 most variable genes were used as input for PCA. Principal components were selected based on an elbow plot.
  • the significance of the first twenty principal components was evaluated using the Jackstraw test. Based on these results, on the percentage of variance explained by each component and the number of cells in the dataset, the first five principal components were used for clustering and t-Stochastic Neighbor Embedding (tSNE) analysis. Cluster membership was determined using shared nearest neighbor graph embedding and optimization of the Louvain algorithm as implemented in the Seurat FindNeighbors and FindClusters functions with a resolution of 0.6. All differentially expressed genes were calculated using the Wilcoxon Rank Sum test as implemented in Seurat.
  • the FindAUMarkers function was used to test genes showing a minimum log fold change of 0.25 (for the whole brain vascular and microglia single-cell experiments) or 0.1 (for the cortical microglia single-cell experiment) in each cluster versus all other clusters and expressed in a minimum of 30% of cells in the cluster of interest.
  • cluster annotation was performed manually based on the expression of canonical markers and clusters collapsed based on common cell types.
  • a final number of 7286 cells, including 2625 microglia, 1958 capillary blood endothelial cells (BECs), 1412 venous BECs and 545 arterial BECs was obtained in the whole brain vascular and myeloid single-cell dataset obtained using adult 5xFAD mice.
  • a final number of 7739 cells including 2345 microglia, 2934 capillary BECs, 602 venous BECs and 766 arterial BECs, was obtained in the whole brain vascular and myeloid single-cell dataset obtained using aged APPswe mice.
  • small populations of potentially contaminating oligodendrocyte precursor cells, oligodendrocytes, neurons and astrocytes were identified and removed based on the expression levels of Cspg4, Mbp, Cam2ka, and Gfap genes, respectively.
  • Cspg4, Mbp, Cam2ka, and Gfap genes were identified and removed based on the expression levels of Cspg4, Mbp, Cam2ka, and Gfap genes, respectively.
  • one population of cells was removed based on cluster markers, leading to a final number of 649 microglia.
  • each cluster was filtered to include genes that had at least 5 transcripts in at least 5 cells, then the top 2,000 highly variable genes were determined and included for further analysis using the SingleCellExperiment modelGeneVar and getTopHVGs functions.
  • observational weights for each gene were calculated using the ZINB-WaVE zinbFit and zinbwave functions. These were then included in the edgeR model, which was created with the glmFit function, by using the glmWeightedF function. Results were then filtered using as threshold a Benjamini-Hochberg adjusted /’-value ⁇ 0.05 (statistically significant).
  • Volcano plots were made with the EnhancedVolcano package. Genes matching this significance threshold were divided on the basis of up- or down-regulation and used as input for GO Analysis using the ClusterProfiler package.
  • the FindMarkers function was used to test all genes expressed in at least 10% of cells within each group /’-values were adjusted using the Bonferroni correction method and genes were considered to be significantly differentially expressed if the adjusted /’-values ⁇ 0.05.
  • RNA-seq data of FECs from diaphragm or from ear skin were included in the heatmap visualizations for reference but were not used for calculation of the average expression. Heatmaps were visualized using the log2 transformed expression values and the pheatmap package.
  • the average normalized expression of each gene was calculated within each microglial cluster (clusters 1-4) and the percentage of AD-associated genes falling within each quantile was determined based on average expression of the gene within the cluster.
  • the union of the sets of AD-associated genes falling within the top 2 nd percentile of highly expressed genes for each cluster was visualized in a heatmap using log2 average normalized expression values.
  • the gene set was determined by ranking genes for that cell type based on average expression across all cells in the dataset rather than by cluster.
  • RNA-seq data sets have been deposited online in the Gene Expression Omnibus (GEO database) under the accession number GSE141917. Previously published RNA-seq data sets can be found under the accession numbers GSE99743 and GSE104181. Code used to analyze the RNA-seq data is available online under GNU General Public license v3.0 at Github under Kipnis Lab / DaMesquita-2019. Custom code used and datasets generated and/or analyzed during the current study are also available from the corresponding authors upon reasonable request.
  • Example 11 Treatment with VEGF-c and aducanumab
  • a human subject is identified as suffering from Alzheimer’s disease.
  • a pharmaceutical composition comprising human VEGF-c and aducanumab is administered monthly to the subject via intravenous infusion. The intravenous administration is repeated monthly for eight months.
  • Amelioration of behavior symptoms of Alzheimer’s disease, including memory loss is observed.
  • Reduction in quantity of density of amyloid beta plaques in the brain of the subject is observed by in vivo magnetic resonance imaging.
  • the method, use, or composition comprises various steps or features that are present as single steps or features (as opposed to multiple steps or features).
  • the method includes a single administration of a flow modulator, or the composition comprises or consists essentially of a flow modulator for single use.
  • the flow modulator may be present in a single dosage unit effective for increasing flow.
  • a composition or use may comprise a single dosage unit of a flow modulator effective for increasing flow as described herein. Multiple features or components are provided in alternate embodiments.
  • the method, composition, or use comprises one or more means for flow modulation.
  • the means comprises a flow modulator.
  • compositions for use in the method are expressly contemplated, uses of compositions in the method, and, as applicable, methods of making a medicament for use in the method are also expressly contemplated.
  • flow modulators for use in the corresponding method are also contemplated, as are uses of a flow modulator in increasing flow according to the method, as are methods of making a medicament comprising the flow modulator for use in increasing flow.
  • a system having at least one of A, B, or C would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”
  • a range includes each individual member.
  • a group having 1-3 cells refers to groups having 1, 2, or 3 cells.
  • a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

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Abstract

Dans certains modes de réalisation, l'invention concerne des procédés, des compositions et des utilisations pour moduler des vaisseaux lymphatiques du système nerveux central. Dans certains modes de réalisation, des procédés, des compositions ou des utilisations pour traiter, prévenir ou soulager des symptômes d'une maladie neurologique comprennent l'augmentation du flux par l'intermédiaire de vaisseaux lymphatiques méningés.
EP21706437.7A 2020-01-24 2021-01-25 Modulation de vaisseaux lymphatiques dans une maladie neurologique Pending EP4093426A1 (fr)

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PCT/US2021/014921 WO2021151079A1 (fr) 2020-01-24 2021-01-25 Modulation de vaisseaux lymphatiques dans une maladie neurologique

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